The Renin-Angiotensin System: Current Research Progress in The Pancreas: The RAS in the Pancreas [1 ed.] 9048190592, 9789048190591, 9789048190607

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The Renin-Angiotensin System: Current Research Progress in The Pancreas

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Editorial Board: IRUN R. COHEN, The Weizmann Institute of Science ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research JOHN D. LAMBRIS, University of Pennsylvania RODOLFO PAOLETTI, University of Milan

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HOX GENES: STUDIES FROM THE 20TH TO THE 21ST CENTURY Edited by Jean S. Deutsch

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THE RENIN-ANGIOTENSIN SYSTEM: CURRENT RESEARCH PROGRESS IN THE PANCREAS Edited by Po Sing Leung

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Po Sing Leung

The Renin-Angiotensin System: Current Research Progress in The Pancreas The RAS in the Pancreas

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Po Sing Leung, PhD School of Biomedical Sciences Faculty of Medicine The Chinese University of Hong Kong Shatin Hong Kong China [email protected]

ISSN 0065-2598 ISBN 978-90-481-9059-1 e-ISBN 978-90-481-9060-7 DOI 10.1007/978-90-481-9060-7 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2010933093 © Springer Science+Business Media B.V. 2010 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

To my wife, Wan Chun Hu and my daughter, Choy May Leung and my son Ho Yan Leung

Preface

The human pancreas consists of two organs in one: the exocrine gland made up of pancreatic acinar cells and duct cells that produce digestive enzymes and sodium bicarbonate, respectively; the endocrine gland made up of four islet cells, namely alpha-, beta-, delta- and PP-cells that produce glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. While the physiological role of exocrine pancreas is to secrete digestive enzyme responsible for our normal digestion, absorption and assimilation of nutrients, the endocrine pancreas is to secrete islet peptide hormones maintaining our glucose homeostasis. The pancreatic functions are finely regulated by neurocrine, endocrine, paracrine and/or intracrine mechanisms. Thus, dysregulation of these pathways should have significant impacts on our health and disease. Nevertheless, the underlying mechanisms by which pancreatic functions are regulated remain poorly understood. Recent basic science and clinical studies confirm myriad physiological and pathophysiological roles of the tissue renin-angiotensin systems (RAS). Of particular interest is the recent identification of a local and functional RAS in the pancreas, which influences both its exocrine and endocrine function. Its role in the pathogenesis of pancreatic diseases including diabetes and pancreatitis is increasingly recognized, as is the therapeutic potential of RAS antagonism: RAS blockade limits disease progression of type 2 diabetes mellitus and impaired glucose tolerance, and may also protect against pancreatic inflammation. To date, no single book has focused exclusively on the pancreatic RAS, and the author believes that such a publication is long overdue. This volume is the response, being an effort to draw together the current state of knowledge in the field, from the basic research to the bedside. It is our hope that the contents will excite and inform not just experts in the field, but those working in parallel disciplines and areas. Finally, I would like to take this opportunity to express my sincere gratitude to Dr. Max Haring, Publishing Editor and Miss Marlies Vlot, Senior Assistant of Springer for their continued support and encouragement. I also express my appreciation to my graduate students, Raymond Leung, Qianni Cheng, Fanny Ma, Cynthia Lau, Ada Suen and Yuk Cheung Chan as well as Eric Wong, my technician, for their assistance. At last but not the least, I greatly appreciate the financial support

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for my projects provided by Research Grants Council of Hong Kong, Merck Sharp & Dohme, Novartis and Boehringer Ingelheim. Without all of the above generous support, my book could not have been published as it stands. Hong Kong, China December 2009

Po Sing Leung, PhD

Prologue

In 1898, Robert Tiegerstedt first described renin as a “pressor substance”. Within 80 years, the role of the circulating endocrine renin-angiotensin system (RAS) in circulatory homeostasis was well described and, by 1990, angiotensin converting enzyme inhibition was transforming the pharmacological management of cardiac failure and hypertension. It was at much the same time, however, that the existence of local tissue and cellular RAS was beginning to be described. These were exciting times: before long, we knew that local RAS played a role in cardiac growth, tissue fibrosis and wound healing, cellular inflammatory responses, transduction of load in skeletal muscle, cerebral function, insulin sensitivity, adipocyte growth and differentiation. . . . The list went on and on. Some of these findings were pursued: they were revealed while the various RAS antagonists (ACE inhibitors and AT1 receptor antagonists) still had sufficient remaining patent life to offer commercial incentive to the manufacturing companies. Just as such interest was waning, however, Po Sing Leung and others began to describe a pancreatic RAS—and one with extraordinary possible implications to disease pathogenesis and management. Their findings had implications for the prevention and treatment of diabetes, and also of other “orphan diseases” fore which no other specific therapies existed (such as pancreatitis). It is a shame that such findings appeared as commercial interest was declining. Nonetheless, these findings remain important- and new life may be breathed into their pursuit by the recent launch of direct renin inhibition. This volume offers a welcome summary of the “state of the art” of this field. I, for one, earnestly hope that it encourages some to take an active interest. There is much to be done and much potential benefit yet for the prevention and treatment of pancreatic endocrine and exocrine disease. Neither would it surprise if local RAS were found to play a powerful role in endocrine regulation elsewhere in the body. Such, in general, is the way of nature. . . London, UK December 2009

Hugh Montgomery, MD

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Prologue

Just when we thought we knew everything about the renin-angiotensin system (RAS), a whole new phase of discovery has emerged. Through his work and writings, Po Sing Leung points the way for many of us to become more engaged and open-minded, particularly in pushing conceptual development in this field. This book provides rich insights into how the RAS regulates both development and regulation of endocrine and exocrine functions of the pancreas. By doing so, it helps unravel the intricacies of process and mechanism of a complex system that impacts health and disease. These paradigms are not likely to be unique to the pancreas, but also extend to other organ systems. For this reason, this important book will be of interest to many disciplines and will serve as a foundation and catalyst for future discovery in this field. Chicago, IL December 2009

Eugene B. Chang, MD

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Contents

Part I

The Pancreas

1 Overview of the Pancreas . . . . . . . . . . . . . . . . 1.1 Structure of the Pancreas . . . . . . . . . . . . . . 1.2 Development of the Pancreas . . . . . . . . . . . . 1.3 Molecular Hierarchy of the Pancreatic Development References . . . . . . . . . . . . . . . . . . . . . . . . .

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2 Physiology of the Pancreas . . . . . . 2.1 Exocrine–Endocrine Axis . . . . 2.2 Pancreatic Acinar Cells . . . . . 2.3 Pancreatic Duct Cells . . . . . . 2.4 Pancreatic Islet Cells . . . . . . . 2.4.1 Beta-Cells . . . . . . . . 2.4.2 Alpha-Cells . . . . . . . 2.4.3 Delta-Cells and PP-Cells . References . . . . . . . . . . . . . . .

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3 Common Pancreatic Disease . . . . . . . . . . . . . . . . . 3.1 Pancreatitis . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1 Etiology and Prognosis . . . . . . . . . . . . . 3.1.2 Treatments . . . . . . . . . . . . . . . . . . . . 3.2 Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Etiology and Prognosis . . . . . . . . . . . . . 3.2.2 Treatments . . . . . . . . . . . . . . . . . . . . 3.3 Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . 3.3.1 Etiology and Prognosis . . . . . . . . . . . . . 3.3.2 Treatments . . . . . . . . . . . . . . . . . . . . 3.3.3 Alternative Approach Using Traditional Chinese Medicine . . . . . . . . . . . . . . . . . . . . . 3.4 Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . 3.4.1 Etiology and Prognosis . . . . . . . . . . . . . 3.4.2 Treatments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Part II

Contents

The Renin-Angiotensin System (RAS)

4 Circulating RAS . . . . . . . . . . . . . 4.1 Definition of Circulating RAS . . . . 4.2 Angiotensinogen . . . . . . . . . . . 4.3 Angiotensin-Generating Enzymes . . 4.4 Angiotensin and (Pro)renin Receptors References . . . . . . . . . . . . . . . . .

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5 Local RAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Definition of the Local RAS . . . . . . . . . . . . . . . . 5.2 Local RAS in Carotid Body . . . . . . . . . . . . . . . . 5.2.1 Expression and Function of Carotid Body RAS . . 5.2.2 Carotid Body RAS and Congestive Heart Failure . 5.3 Local RAS in Liver . . . . . . . . . . . . . . . . . . . . 5.3.1 RAS and Liver Function . . . . . . . . . . . . . . 5.3.2 Interaction Between Hepatic RAS and Vitamin D in T2DM . . . . . . . . . . . . . . . . . . . . . . 5.4 Local RAS in Intestine . . . . . . . . . . . . . . . . . . . 5.4.1 Expression and Function of an Enterocyte RAS . . 5.4.2 Enterocyte RAS and Diabetes Mellitus . . . . . . 5.4.3 ACE2-Angiotensin (1–7)-Mas Receptor Axis and Intestinal Glucose Uptake . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6 Pancreatic RAS . . . . 6.1 Acinar Cell RAS . 6.2 Ductal Cell RAS . 6.3 Stellate Cell RAS 6.4 Islet Cell RAS . . References . . . . . . .

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7 Basic Techniques for Pancreatic Research . . . . . 7.1 Cell Models . . . . . . . . . . . . . . . . . . . 7.2 Animal Models . . . . . . . . . . . . . . . . . . 7.2.1 Animal Models of T1DM . . . . . . . . 7.2.2 Animal Models of T2DM . . . . . . . . 7.2.3 Animal Models of Pancreatitis . . . . . . 7.2.4 Animal Models of Pancreatic Cancer . . 7.3 Islet and Acinar Cell Isolation . . . . . . . . . . 7.4 Islet Transplantation . . . . . . . . . . . . . . . 7.4.1 Animal Models of Islet Transplantation . 7.4.2 Recent Advances in Islet Transplantation 7.5 Expression and Functional Studies . . . . . . . 7.5.1 Gene expression studies . . . . . . . . .

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Part III Research Progress of the RAS in Pancreas

Contents

7.5.2 7.5.3 7.5.4 References

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Protein Expression Studies Transfection . . . . . . . . Functional Studies . . . . . . . . . . . . . . . . . . . . .

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8 Current Research of the RAS in Diabetes Mellitus . . . . . . 8.1 Basic Studies of the RAS in T2DM . . . . . . . . . . . . . 8.1.1 RAS Blockade Studies in Animal Models of T2DM 8.1.2 RAS Blockade-Induced Protective Mechanism . . . 8.2 Clinical Studies of the RAS in T2DM . . . . . . . . . . . . 8.2.1 Recent Clinical Trials on RAS Blockers . . . . . . . 8.2.2 DREAM Trial and Its Implications . . . . . . . . . 8.3 Current Research on the RAS-Vitamin D-T2DM Axis . . . 8.3.1 Vitamin D and RAS in T2DM . . . . . . . . . . . . 8.3.2 Role of Vitamin D in Modulating Islet RAS Expression and Function . . . . . . . . . . . . . . . 8.3.3 Expression of Islet RAS in VDR Knockout Mice . . 8.4 Current Research on the RAS-GLP1-T2DM Axis . . . . . 8.4.1 Role of DPP-IV Inhibition in T2DM . . . . . . . . 8.4.2 Interaction of Islet RAS and GLP-1 . . . . . . . . . 8.4.3 Potential Role of Vitamin D in Modulating Islet RAS and GLP-1 . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9 Current Research Concerning the RAS in Pancreatic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Source of Pancreatic Stem Cells . . . . . . . . . . . . 9.2 Current Research on Pancreatic Stem Cells . . . . . . 9.3 Current Research of the RAS on Stem Cells . . . . . 9.3.1 The RAS and ESCs . . . . . . . . . . . . . . 9.3.2 The RAS and MSCs . . . . . . . . . . . . . . 9.3.3 The Vascular RAS and Erythropoiesis . . . . . 9.3.4 The RAS and Fetal Tissues . . . . . . . . . . 9.4 Current Research on the RAS in Pancreatic Stem Cells 9.4.1 Limitations and Future Directions . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . 10

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Current Research of the RAS in Pancreatitis and Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Basic Studies of the RAS in Pancreatitis . . . . . . . . . 10.1.1 Evidence of a Local RAS and Its Roles in Pancreatitis . . . . . . . . . . . . . . . . . . . 10.1.2 Potential Mechanisms of RAS-Mediated Oxidative Stress and Blood Flow in Pancreatitis . 10.1.3 The RAS and Cytokine-Mediated Systemic Inflammation in Pancreatitis . . . . . . . . . . . .

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10.2 Current Research of the RAS on Pancreatitis In Vivo . 10.2.1 Effects of RAS Blockade in Rat Model of Caerulein-Induced AP . . . . . . . . . . . 10.2.2 Effects of RAS Blockade in Rat Model of Obstructive AP . . . . . . . . . . . . . . . 10.3 Current Research of the RAS on Pancreatitis In Vitro 10.3.1 Effects of Angiotensin II on ERK and CREB-Mediated IL6 Expression . . . . . 10.3.2 Potential Crosstalk Between Angiotensin II-Induced ROS-Mediated ERK and NFκB . . 10.4 Current Research of the RAS in Pancreatic Cancer . . 10.4.1 Expression and Regulation of the RAS Components in Tumours . . . . . . . . . . . . 10.4.2 RAS Blockade and Pancreatic Cancers . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Part I

The Pancreas

Chapter 1

Overview of the Pancreas

1.1 Structure of the Pancreas Structurally speaking, the human pancreas consists of two organs in one: an exocrine gland and an endocrine gland. The exocrine gland is made up of pancreatic acinar cells and duct cells that produce digestive enzymes and sodium bicarbonate, respectively. The primary function of the exocrine pancreas is to secrete the digestive enzymes responsible for normal digestion and absorption of daily foodstuffs, and finally assimilation of nutrients into our body. The endocrine gland, meanwhile, is made up of five types of secretory islet cells and secretes peptide hormones for the maintenance of glucose homeostasis. The pancreatic secretory functions are finely regulated by neurocrine, endocrine, and paracrine as well as intracrine mechanisms. In view of this fact, inappropriate activation or inactivation of the pathways mediating the pancreas’s fine regulatory mechanisms has considerable impacts on health and disease. Nevertheless, the underlying local mechanisms by which pancreatic function and dysfunction are regulated remain poorly understood. The human pancreas is a retroperitoneal organ of the upper abdomen that, on average, weighs in the range of 100–150 g and measures 15–25 cm in length. It secretes about a kilogram of pancreatic juice daily into the duodenum via the ampulla of Vater where the main pancreatic duct coalesces with the common bile duct. Anatomically, the pancreas is connected with other abdominal organs including the spleen, stomach, duodenum and colon. The pancreas is structurally divided into three parts, termed the head, body and tail. The head region of the pancreas is relatively flat and situated within the first loop of the duodenum. The tail region is in close vicinity to the hilum of the spleen; it is the only part of the pancreas that contains pancreatic polypeptide (PP) cells that produce the peptide hormone PP (see Section 2.4, Chapter 2). The body region of the pancreas has a shape that resembles a prism. The blood supply of the pancreas depends upon several major arteries, namely the inferior pancreaticoduodenal artery, the superior pancreaticoduodenal artery, and the splenic artery. Pancreatic removal of metabolites and hormone release are mediated via the pancreaticoduodenal vein and the pancreatic vein, respectively. The pancreas is highly innervated by the pancreatic plexus, celiac ganglia, and vagus nerve. For additional information of the anatomy, function and disease of the P.S. Leung, The Renin-Angiotensin System: Current Research Progress in The Pancreas, Advances in Experimental Medicine and Biology 690, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9060-7_1, 

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1 Overview of the Pancreas

pancreas, please refer to an updated and integrated textbook of basic and clinical aspect of the pancreas (Beger, 2008). The endocrine and exocrine roles of the pancreas, though distinct, have closely interrelated physiological functions. The pancreas can be considered as four structurally distinct components: the exocrine pancreas, consisting principally of acinar cells and duct cells; the endocrine pancreas, the site of the islet cells; the blood vessels; and the extracellular space. The exocrine portion, whose major function is to produce digestive enzyme and sodium bicarbonate secretion, accounts for most, if not all, of the cellular mass of the pancreas (i.e. approximately 80% by volume). The exocrine pancreas architecture is characterized by a blind-ended ductal system, structurally analogous to a massive bunch of grapes, such that each acinus equivalent to a grape is bounded by adjacent acinar cells that secrete pancreatic juice enzymes into a blind-ended tubule or called stem. The acini are grouped into lobules with a branched network of tubules. Each acinus is composed of highly orientated, pyramidal-shaped acinar cells, with their apical membranes lining a central lumen known as an intercellular canaliculus and their basolateral membranes forming the acinar periphery. The acinar cell secretions flow into the intercellular canaliculi, which consist of ductal epithelial cells, also known as duct cells, which secrete bicarbonate, mainly in the form of sodium bicarbonate. The tiny intercalated ducts that drain the acini converge into larger intralobular ducts, which then converge into major extralobular duct. The extralobular ducts finally converge into a main collecting duct, which joins with the common bile duct before the pancreatic juice enters the duodenum (see review by Leung & Ip, 2006). For the purpose of this chapter, a highly schematic illustration of the ductal and acinar cells of the exocrine pancreas is summarized and presented in Fig. 1.1. On the other hand, the endocrine pancreas is composed of clusters of cells formerly known as the islets of Langerhans, or more simply termed pancreatic islets, to which the secretion of a number of pancreatic peptide hormones for glucose homeostasis is attributed. The endocrine pancreas is highly vascularized and morphologically distinct from its exocrine counterpart. The islets are structurally arranged in spherical shape, and account for 1–2% by volume of the total pancreatic mass. There are five major cells which constitute the islet, namely the alpha cells (α-cells), beta cells (β-cells), delta cells (δ-cells), PP cells (also known as F-cells) and, to some extent, the epsilon cells (ε-cells), which are responsible for producing glucagon, insulin, somatostatin, pancreatic polypeptide, and ghrelin, respectively (see review by Kulkarni, 2004). The secretory functions of the islet cells are modulated by signalling factors from inside and outside the pancreas. These cells can communicate with one another and influence one another’s secretion. Islet cell communication modalities include, but are not limited to, humoral communication, cell–cell communication, and neural communications. In addition, there is intimate contact between the endocrine and exocrine pancreas by means of an insulin-acinar axis (Williams & Goldfine, 1985). The physiological function of the endocrine cells and their regulatory mechanisms will be discussed further in Chapter 2. In summary, a schematic illustration of the cell types and structure of the insulino-acinar portal system of the endocrine pancreas are illustrated in Fig. 1.2.

1.1

Structure of the Pancreas

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Fig. 1.1 A highly schematic diagram showing the structure and relationship of the ductal epithelial and acinar cells of the exocrine pancreas

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1 Overview of the Pancreas

Fig. 1.2 A highly schematic diagram showing the different cell types within the islet of Langerhans and the structure of insulo-acinar portal system of the endocrine pancreas

1.2 Development of the Pancreas Our understanding of human pancreatic development is based largely on murine model studies as the patterns of its developmental stages are well conserved between humans and mice. The developmental processes are generalized into stages of organ specification, expansion, differentiation, and maintenance, though these events overlap in time (Murtaugh, 2008). On the whole, pancreas development has been described as an epithelial-mesenchymal process wherein signals from the mesenchymal tissues govern organogenesis. Embryologically, the development of the pancreas begins with two endodermlined primordial buds, or outpouchings, of the duodenum during the fourth week of human gestation. The dorsal bud extends into the dorsal mesentery to eventually form the head, body and tail of the pancreas. The ventral bud arises immediately adjacent to the hepatic diverticulum and is associated with the biliary system. Hence, the pancreas originates from two primordial buds of the primitive duodenum and is first apparent at 4 weeks gestation in humans and around embryonic day E9.5 in mice. The developmental programme of the two buds is slightly asynchronous

1.2

Development of the Pancreas

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because they receive distinct signals from the surrounding tissue context. The dorsal pancreatic bud develops in close proximity to the notochord, while the ventral bud develops in close association with the liver and under the control of signals from the overlying cardiogenic mesenchyme. The dorsal pancreatic bud grows more rapidly than the ventral pancreas, and extends into the dorsal mesentery by 6 weeks gestation. As the stomach and the duodenum rotate, the ventral and dorsal pancreatic buds fuse with the ventral bud, giving rise to the posterior part of the head and the dorsal bud, thereby forming the remainder of the organ. This fusion occurs during the seventh week of gestation in humans and around E12–E13 in mice (Suen, 2007). The dual origin of the organ is responsible for regional differences in the distribution of islet cells in the adult: the dorsal pancreas is rich in glucagon-containing islets while the ventral portion contains PP-rich islets and few glucagon-producing cells. Fusion of the ventral duct with the dorsal duct results in the formation of the main pancreatic duct, termed the duct of Wirsung, which runs through the entire pancreas. The proximal end of the dorsal pancreatic duct usually does not communicate with the main duct, but rather forms the accessory pancreatic duct (Bonner-Weir & Smith, 1994). The pancreas at later stages undergoes a process of rapid branching morphogenesis. The mechanisms of these highly dynamic processes remained poorly understood until a recent study using real-time imaging reported visualization of the budding and branching process (Puri & Hebrok, 2007). In mouse, two temporal waves of endocrine differentiation, termed the first and second transitions, have been observed. The first transition occurs between E9.5 and E12.5, and involves a change in shape of the pancreatic domain and the generation of early endocrine cells with co-expression of several pancreatic hormones. The second transition occurs between E12.5 and birth and encompasses a massive endocrine differentiation and major amplifications of hormone-expressing cell numbers (Pictet et al., 1972). The mature exocrine marker amylase is first detected at E13.5. The human equivalents of these transitions have not been described, though a marked transition of the exocrine genes has been observed at 11 weeks gestation (Sarkar et al., 2008). Groups of endocrine cells develop from multipotential stem cells in the ductal epithelium at 9 weeks gestation. The pancreatic acini and the first zymogen granules appear at 12 weeks gestation. Discrete islets of Langerhans can be identified at 12 weeks, and 1 week later, large primitive islet structures expressing all four pancreatic hormones are formed. While the mechanisms of specification and the reciprocal relationships of the four types of endocrine cell (alpha, beta, delta and PP cells) within the human endocrine pancreas are relatively well described during development, ghrelin-secreting epsilon cells (ε-cells) remain poorly understood; in this regard, scattered ghrelin-positve ε-cells have been observed during this period; they start to aggregate and localize around the developing islets in subsequent gestational weeks (Andralojc et al., 2009). These data have shown that ghrelin-secreting ε-cells have an ontogenetic and morphogenetic pattern that is distinct from that of apha and beta cells during development. Developing blood vessels start to penetrate into the primitive islets at 14 weeks gestation. The islets continue to increase in size through the second and third trimester (Piper et al., 2004). Most of the islet cells develop within the tail of the pancreas and in the dorsal pancreas.

8

1 Overview of the Pancreas

Fig. 1.3 A schematic illustration showing the different stages of progressive pancreatic development along with its corresponding gut and hepatic systems (modified from Beger, 2008)

The first cells to produce granules are the α-cells, followed soon thereafter by the β- and δ-cells. Complete maturation of the pancreatic gland does not occur until sometime after birth. Lineage tracing experiments have provided direct evidence that exocrine, endocrine islet, and duct progenitors are committed at mid-gestation and are from different lineages (Gu et al., 2002). The islet cells appear to arise from stem cells that become visible in pancreatic ducts during the third month of gestation. Islet cells migrate away from the ducts in which they arose, and move into the interlobular connective tissue. Mature islet morphology is established before birth (Kulkarni, 2004). Intriguingly, it has been more recently proposed that islet formation occurs by a process of fission following contiguous endocrine cell proliferation, rather than by local aggregation or fusion of isolated beta-cells and islets (Miller et al., 2009). Taken together, the various development stages from embryonic ventral and dorsal buds to complete formation of the pancreas and its ductal system are summarized in Fig. 1.3.

1.3 Molecular Hierarchy of the Pancreatic Development Involvement of Wnt/β-catenin signalling in initiating pancreas development has been widely described. Inhibition of the Wnt/β-catenin pathway appears to be essential for the initial pancreatic specification during expansion of the pancreatic

1.3

Molecular Hierarchy of the Pancreatic Development

9

epithelium; such signalling mechanisms become necessary later in development (Murtaugh, 2008; Mclin et al., 2007). Pancreas generation from the gut endoderm is controlled by signals generated in the adjacent notochord. These include, among others, transforming growth factor-beta (TGFβ), activin β, and basic fibroblast growth factor (bFGF, also known as FGF2). Deficiencies in expression of these growth factors have been shown to disrupt pancreatic morphology, with epithelial structures becoming interspersed in the surrounding stroma tissue (Dichmann et al., 2003). These molecules act via repression of the expression of sonic hedgehog (Shh) in the dorsal region of the gut, the region that can subsequently form the pancreatic epithelium in the presence of pancreatic duodenal homeobox 1 (PDX-1) (Hebrok et al., 2000). Of note, the hedgehog signalling often regulates tissue morphogenesis in a dose-dependent manner. Inhibition of Shh signalling is essential in the initiation of pancreatic development in rodents. Ectopic expression of Shh can even result in transformation of pancreatic mesenchyme into duodenal mesoderm (Kawahira et al., 2003). Yet in other species, like zebrafish, Shh appears to be indispensible for proper pancreatic development (Dilorio et al., 2002). The molecular hierarchy of pancreatic development is temporally and spatially controlled by an array of transcription factors. Their regulation determines the initial budding of the organ as well as cell fates towards the endocrine or exocrine lineage. PDX-1 (also known as IPF-1, STF-1, IDX-1 and IUF-1) plays a crucial role in the growth and differentiation of the pancreatic buds (Kaneto et al., 2008). Its expression is maintained in pancreatic precursors and becomes restricted to insulin-producing β-cells as the pancreas matures. Mice lacking this gene fail to form a pancreas. The pancreatic buds in these embryos and the dorsal bud undergo limited proliferation and outgrowth. These observations indicate that although PDX-1 defines a distinct compartment of the endodermal foregut that specifies the dorsal and ventral pancreas, additional genes must be involved in the early stages of pancreatic specification and bud formation. HNF3β (also known as Foxa2) may be involved in this early stage, as it has been shown to regulate PDX-1 promoter activity (Ben-Shushan et al., 2001). Other genes expressed early in pancreatic development, including the homeobox gene hlxb9 (encoding Hb9) and LIM-domain protein Isl1, are involved in the dorsal mesenchymal but not the ventral pancreatic programme. Following formation of the pancreatic buds and concomitant with the growth of the branching epithelial network, a second phase of transcription factor expression occurs. These factors include the basic helix-loop-helix (bHLH) proteins Ptf1/p48 (p48) and ngn3—factors that appear to control commitment towards an endocrine or exocrine lineage. Ptf1/p48 is the DNA-binding subunit of the hetero-oligomeric transcription factor Ptf1, which controls expression of genes in the exocrine pancreas (Krapp et al., 1998). Ptf1 is also required in the early patterning of undifferentiated epithelium; it is later restricted to pancreatic acinar cells. A recent study reported that an enhancer located downstream of the Ptf1 gene established an autoregulatory loop with the gene to reinforce and maintain its expression in mature acinar cells (Masui et al., 2008). Neurogenin 3 (Ngn3) is a bHLH protein that is transiently expressed in the developing pancreas and is critical to the specification of all four types of islet

10

1 Overview of the Pancreas

endocrine cells. It is not present in mature islets. A recent study has revealed biphasic Ngn3 expression in mouse embryonic pancreas. The two waves of expression correlated exactly with the first transition and in the precedence of the second transition of endocrine differentiation (Villasenor et al., 2008). There is strong evidence indicating that Ngn3 defines an islet progenitor cell and that Ngn3-positive cells do not develop into duct cells (Gu et al., 2002). Ngn3 was demonstrated to be a determination factor for the four endocrine cells lineage in a study in which mice with disrupted Ngn3 genes all failed to generate any pancreatic endocrine cells and died postnatally from diabetes (Gradwohl et al., 2000). The expression of Ngn3 is regulated by Delta/Notch signalling on adjacent epithelial cells. Notch

Fig. 1.4 An easy-to-follow schematic representation showing an array of proposed transcription factors that are critical in controlling development of the exocrine and endocrine cell lineage in the pancreas (redrawn from Samson & Chan, 2006)

References

11

is a transmembrane receptor and Delta is a transmembrane ligand, the expression of which is stimulated by Ngn3. When activated by Delta expressed by a close proximal cell, Notch triggers expression of another bHLH protein, Hes1, that inhibits expression of Ngn3. Cells subjected to this inhibition of Ngn3 expression are driven towards an exocrine fate. Downstream of Ngn3 transcription factors lie a number of late-expressed transcription factors: transient expression of Nkx2.2, Pax4, Nkx6.1 and PDX-1 drives Ngn3-positive cells towards a β-cell phenotype; meanwhile expression of Brn4, Nkx6.2 and Pax6 direct cells towards an α-cell phenotype (Gradwohl et al., 2000). Any alteration in the expression of these regulatory genes will disrupt the balance of pancreatic hormone-expressing cells developed. The homeodomain protein Nkx2.2, in particular, is a critical regulator of islet specification. It has been elegantly shown that the endocrine components in Nkx2.2 mutant mice are replaced predominantly by ghrelin-expressing ε-cells (Prado et al., 2004). α-cells and a small portion of β-cells could be rescued in a Nkx2.2-Engrailed-repressor derivation (Doyle et al., 2007). For additional information about the development of the pancreas, please refer to a recent textbook (Scharfmann & Shield, 2007). Figure 1.4 summarizes a raft of proposed transcription factors that play important roles in governing the differentiation and development of the pancreatic exocrine and endocrine cells in the pancreas.

References Andralojc KM, Mercalli A, Nowak KW, Albarello L, Calcagno R, Luzi L, Bonifacio E, Doglioni C and Piemonti L. Ghrelin-producing epsilon cells in the developing and adult human pancreas. Diabetologia 52:486–493, 2009. Ben-Shushan E, Marshak S, Shoshkes M, Cerasi E and Melloul D. A pancreatic β-cell-specific enhancer in the human Pdx-1 gene is regulated by HNF-3β, HNF-1α and SPs transcription factors. J Biol Chem 276:17533–17540, 2001. Beger HG. The Pancreas: an integrated textbook of basic science, medicine and surgery, 2nd edn. Blackwell, Oxford, 2008. Bonner-Weir S and Smith FE. Islets of Langerhans: morphology and its implications. In CR Rahn and GC Weir (eds), Joslin’s diabetes mellitus, Philadelphia, PA, pp 15–28, 1994. Dichmann DS, Miller CP, Jensen J, Scott Heller R and Serup P. Expression and misexpression of members of the FGF and TGFbeta families of growth factors in the developing mouse pancreas. Dev Dyn 226:663–674, 2003. Dilorio PJ, Moss JB, Sbrogna JL, Karlstrom RO and Moss LG. Sonic hedgehog is required early in pancreatic islet development. Dev Biol 244:75–84, 2002. Doyle MJ, Loomis ZL and Sussel L. Nkx2.2-repressor activity is sufficient to specify alpha-cells and a small number of beta-cells in the pancreatic islet. Development 134:515–523, 2007. Gradwohl G., Dierich A, LeMeur M and Guillemot F. Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Dev Biol 97:1607–1611, 2000. Gu G, Dubauskaite J and Melton DA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129:2447-57, 2002. Hebrok M, Kim SK, St Jacques B, McMahon AP and Melton DA. Regulation of pancreas development by hedgehog signaling. Development 127:4905–4913, 2000. Kaneto H, Matsuoka TA, Miyatsuka T, Kawamori D, Katakami N, Yamasaki Y and Matsuhisa M. PDX-1 functions as a master factor in the pancreas. Front Biosci 13:6406–6420, 2008.

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Kawahira H, Ma NH, Tzanakakis ES, McMahon AP, Chuang PT and Hebrok M. Combined activities of hedgehog signaling inhibitors regulate pancreas development. Development 130:4871–4879, 2003. Krapp A, Knofler M, Ledermann B, Burki K, Berney C, Zoerkler N, Hagenbuchle O and Wellauer PK. The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas. Genes Dev 12:3752–3763, 1998. Kulkarni RN. The islet β-cell. Int J Biochem Cell Biol 36:365–371, 2004. Leung PS and Ip SP. Pancreatic acinar cell: its role in acute pancreatitis. Int J Biochem Cell Biol 38:1024–1030, 2006. Masui T, Swift GH, Hale MA, Meredith DM, Johnson JE and Macdonald RJ. Transcriptional autoregulation controls pancreatic Ptf1a expression during development and adulthood. Mol Cell Biol 28:5458–5468, 2008. McLin VA, Rankin SA and Zorn AM. Repression of Wnt/beta-catenin signaling in the anterior endoderm is essential for liver and pancreas development. Development 134:2207–2217, 2007. Miller K, Kim A, Kilimnik G, Jo J, Moka U Periwal V and Hara M. Islet formation during the neonatal development in mice. PLoS One 4:e7739, 2009. Murtaugh LC. The what, where, when and how of Wnt/beta-catenin signaling in pancreas development. Organogenesis 4:81–86, 2008. Pictet RL, Clark WR, Williams RH and Rutter WJ. An ultrastructural analysis of the developing embryonic pancreas. Dev Biol 29:436–467, 1972. Piper K, Brickwood S, Turnpenny LW, Cameron IT, Ball SG, Wilson DI and Hanley NA. Beta cell differentiation during early human pancreas development. J Endocrinol 181:11–23, 2004. Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B and Sussel L. Ghrelin cells replace insulinproducing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci U S A 101:2924–2929, 2004. Puri S and Hebrok M. Dynamics of embryonic pancreas development using real-time imaging. Dev Biol 306:82–93, 2007. Samson SL and Chan L. Gene therapy for diabetes: reinventing the islet. Trends Endocrinol Metab 17:92–100, 2006. Sarkar SA, Kobberup S, Wong R, Lopez AD, Quayum N, Still T, Kutchma A, Jensen JN, Gianani R, Beattie GM, Jensen J, Hayek A and Hutton JC. Global gene expression profiling and histochemical analysis of the developing human fetal pancreas. Diabetologia 51:285–297, 2008. Scharfmann R and Shield JPH. Development of the pancreas and neonatal diabetes. Karger, Basel, 2007. Suen PM. Isolation, characterization and differentiation of pancreatic progenitor cells from human fetal pancreas. Ph.D. Thesis, The Chinese University of Hong Kong, 2007. Villasenor A, Chong DC and Cleaver O. Biphasic Ngn3 expression in the developing pancreas. Dev Dyn 237:3270–3279, 2008. Williams JA and Goldfine ID. The insulin-pancreatic acinar axis. Diabetes 34:980–986, 1985.

Chapter 2

Physiology of the Pancreas

2.1 Exocrine–Endocrine Axis The pancreas plays a central role in digestion and absorption as well as utilization and storage of energy substrates. As described in Chapter 1, it consists of two structurally distinct but functionally integrated glandular systems, namely the exocrine and endocrine pancreas, both of which arise from an outgrowth of the primitive gut. Secretion by the exocrine pancreas is modulated by neural and hormonal signals, particularly in the form of numerous gastrointestinal peptide hormones (Chey & Chang, 2001). Due to the lack of basal membranes or compartmentalization capsules for different cell types in the pancreas, the islets cells are interspersed within the exocrine acini. Acini located near islets, called peri-insular acini, are composed of larger sized cells possessing larger nuclei and more abundant zymogen granules than acini removed from islets, called tele-insular acini. Some secretory products of the islet cells, such as insulin, interact directly with acinar cells and thereby regulate acinar function (Murakami et al., 1992). The exclusive morphology of the peri-insular acini is reflected in the presence of high insulin concentrations in the region (von Schönfeld et al., 1994). It has been reported that diabetes reduces pancreatic exocrine function (Chey et al., 1963), thus suggesting a close interaction between the endocrine and exocrine components of the pancreas. A novel concept of an insulin-acinar axis was proposed in 1985; it was suggested to be involved in the islet-acinar portal system and to participate in physiological regulation of acinar cell function by islet peptides (Williams & Goldfine, 1985). Pancreatic intralobular arteries branch into the islets via the vas afferents, which in turn divide into capillary glomerulus structures within the islets. The efferent vessels protrude into the surrounding exocrine pancreas to form the insulo-acinar portal system. The peri-insular acinar is hence particularly exposed to high concentrations of islet hormones in close proximity. All the efferent islet blood flows into the acinar capillary before leaving the pancreas; in doing so, no blood from the intralobular islets drains directly into veins without passing through the exocrine portion of the pancreas. Other peptide hormones produced by the endocrine pancreas, such as somatostatin, glucagon, pancreatic polypeptide (PP) and ghrelin, contribute to the regulation of pancreatic enzyme synthesis, transport and secretion, as well as to the growth of acinar cells. To better help understand this P.S. Leung, The Renin-Angiotensin System: Current Research Progress in The Pancreas, Advances in Experimental Medicine and Biology 690, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9060-7_2, 

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Fig. 2.1 A schematic presentation of the insulo-acinar portal system. In this system, islet and acinar cells are arranged in such an apposition that they are exposed to close contact via islet peptide hormones thus rendering exocrine and endocrine interaction to influence acinar cell function

notion, an illustration of exocrine–endocrine interactions based on the insulo-acinar portal system is shown in Fig. 2.1. The interaction of insulo-acinar axis can be exemplified by its effects on the exocrine–endocrine functions. In this regard, the regulation of pancreatic exocrine secretion depends on the peptide hormone insulin released by the pancreatic islet β-cells. It is known that infusion of glucose into perfused rat pancreas has been shown to induce release of endogenous insulin, and thereby enhance pancreatic exocrine secretion in response to the peptide hormone CCK or cholecystokinin (Saito et al., 1980). A significant increase in pancreatic secretion was also observed in the presence of exogenous insulin (Patel et al., 2004). Interestingly, pretreatment with the muscarinic cholinergic receptor antagonist atropine abolished insulininduced increases in pancreatic secretion in hyperglycemic rats (Patel et al., 2004). This observation implies that pancreatic secretion is stimulated by vagal cholinergic activation, which is evoked by hypoglycemia. Exogenous insulin has also been reported to inhibit pancreatic exocrine bicarbonate secretion stimulated by secretin in dogs (Berry & Fink, 1996; Howard-McNatt et al., 2002). However, hyperinsulinemia alone did not inhibit bicarbonate secretion. In contrast, hyperinsulinemia with normoglycemia reduces the bicarbonate secretion (Simon et al., 2002), demonstrating that the inhibitory effect of hyperglycemia on pancreatic secretion may be independent of insulin. Several studies have shown that endogenous somatostatin secreted by the pancreatic δ-cells inhibits secretin and CCK release thus pancreatic exocrine secretion (Chey & Chang, 2001). Since somatostatin is present in both the intestinal mucosa

2.2

Pancreatic Acinar Cells

15

and the pancreas, it is hypothesized that mucosal somatostatin may play a role in the regulation of gut hormones. Meanwhile, pancreatic somatostatin regulates pancreatic exocrine secretion directly, in the form of paracrine messengers (Beger et al., 2008). Somatostatin and PP also have suppressive roles in the insulo-acinar axis (Nakagawa et al., 1993). In contrast, the effect of glucagon on the exocrine secretion remains rather controversial. Intravenous injection of glucagon transiently can activate then suppress secretin and CCK-stimulated somatostatin, followed by an increase in circulating somatostatin in dogs (Horiuchi et al., 1993), suggesting that glucagon-mediated regulation of exocrine secretion may occur indirectly. Functional interactions have been found between the exocrine and endocrine pancreas. For example, many diabetic patients experience changes in pancreatic exocrine function (Chey et al., 1963). However, the impaired exocrine function in these patients may not be directly linked to insulin deficiency since abnormal pancreatic exocrine functions have also been observed in some patients with type 2 diabetes mellitus (Hardt et al., 2000). Insulin deficiency in diabetic patients may be due to the inadequate actions of insulin on the potentiation of secretin and CCKstimulated secretion, induction of amylase gene transcription (Korc et al., 1981), and down-regulation of its own receptors (Okabayashi et al., 1989).

2.2 Pancreatic Acinar Cells The pancreatic acinar cell belongs to the exocrine portion of the pancreas. The pyramid-shaped, serous acinar cell is the dominant cell type in the pancreas, constituting 82% of the total volume of the pancreas (Bolender, 1974). Its major function is to produce, store and secrete three major categories of digestive enzymes: α-amylase, lipase and protease, which are responsible for the digestion of carbohydrates, fats and proteins, respectively. Acinar cells within each acinus are linked by gap junctions. The extensive network of ductal system of the exocrine pancreas (see Fig. 1.1, Chapter 1) allows both chemical and electrical intercellular communications to occur among acinar cells (Iwatsuki & Petersen, 1978). An acinar cell consists of a basally located nucleus, abundant mitochondria, free ribosomes and rough endoplasmic reticulum, and contains a well developed Golgi apparatus. This organelle architecture enables production of abundant secretory granules, called zymogen granules (ZGs), which are composed of a multitude of enzymes and precursor enzymes that are polarized at the apical side of the acinar cells. ZG protease precursors include trypsinogen, chymotrypsinogen and procarboxypeptidases, which are secreted along with the isotonic NaCl mixture called pancreatic juice. Upon its release into the duodenum, inactive trypsinogen is converted into active trypsin by an enzyme called enterokinase, which is anchored to the apical membrane of enterocyte. The duodenally bound enterokinase can specifically cleave 6 amino acids from the N-terminal of trypsinogen to produce a 223-amino acid protein containing trypsin. Positive feedback by trypsin can auto-catalytically further cleave trypsinogen into trypsin and activate other precursor proteases into their respective active forms, such as chymotrypsinogen

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2 Physiology of the Pancreas

into chymotrypsin, procarboxypeptidase into carboxypeptidase and proelastase into elastase. Physiologically speaking, activation of proteases in the duodenum rather than within the pancreas prevents auto-digestion of the pancreas, thus leading to pancreatitis. In addition to proteases, the acinar secretion also contains active digestive enzymes such as pancreatic lipase, amylase and nuclease, as well as other secretory products such as co-lipase, the latter being not a hydrolytic enzyme itself (Beger, 2008). Acinar secretory products need to be directed to the intercellular canaliculi, and the ductal system, and finally into the gut lumen. Ca2+ -activated Cl− channels are found specifically on the apical plasma membrane of acinar cells. Opening of these channels releases Cl– ions into the acinar lumen; the increased negative charge leads to the influx of extracelluar Na+ through tight junctions between acinar cells. NaCl in the acinar lumen then osmotically draws in water and thus constitutes the acinar fluid (Petersen, 2007). There are some basic mechanisms that govern the regulation of pancreatic exocrine secretory functions. While the primary stimulus is the food itself, neural and hormonal systems play a central role in regulating secretion of acinar cells via neurocrine, endocrine and paracrine signalling pathways. The major regulators include the neurocrine molecule acetylcholine (ACh) and the endocrine molecule cholecystokinin (CCK), as well as some paracrine/endocrine molecules such as secretin, vasoactive intestinal polypeptide (VIP) and angiotensin II (Leung & Ip, 2006). These secretagogues act on their respective receptors located at the basolateral plasma membrane of acinar cells, thus evoking downstream signalling transduction pathways mainly in the two forms of intracellular signalling pathways. ACh and CCK exert their effects on the exocrine pancreas by binding their respective muscarinic receptors and CCKB receptors, respectively. Both muscarinic and CCKB receptors in the exocrine pancreas are linked to Gαq G-proteins, use the phospholipase C (PLC)/Ca2+ signal transduction pathway, and lead to increased enzyme secretion from acinar cells. On the other hand, the actions of VIP and secretin are mediated via their respective receptors and both activate Gαs G-proteins which stimulates adenylate cyclase (AC), leading to the production of cAMP and the activation of protein kinase A (PKA). PKA and PKC finally trigger ZG fusion with the apical plasma membrane, leading to the polarized secretion of the digestive enzymes or exocytosis into the acinar lumen (Wäsle & Edwardson, 2002). It is generally accepted that exocytosis is activated by a rise in cytosolic Ca2+ levels. Given that the pancreatic acinar cell is not electrically excitable, though, it cannot rely on the extracellular Ca2+ influx to stimulate enzyme secretion (Petersen, 1992). The Ca2+ concentration required can be achieved from intracellular stores. This concept was well illustrated by experimental data showing that the initial secretory response to either ACh or CCK is independent of extracellular Ca2+ . Meanwhile, prolonged secretion does need a surge in extracellular Ca2+ as intracellular Ca2+ stores are depleted (Petersen & Ueda, 1976). ACh and CCK can evoke various types of Ca2+ signals within acinar cells; the deviation is dependent on the concentration of the secretagogue that the acinar cells encounter (Scheele et al., 1987). Low concentrations of ACh or CCK induce weak local Ca2+ signals near the secretory pole of the acinar cells. In contrast, a high concentration of secretagogue

2.3

Pancreatic Duct Cells

17

Fig. 2.2 Regulated secretion of digestive enzyme from the acinar cell. The pancreatic acinar cell has two common signaling pathways for the exocytosis of secretory zymogen granules into the acinar lumen. They are the ACh and CCK-mediated PKC/Ca2+ pathway and VIP and secretinmediated cAMP/PKA signaling pathways. The angiotensin II-mediated transduction pathway, probably via Ca2+ , has yet to be determined

produces a global Ca2+ wave that spreads over the entire cell (Kasai et al., 1993). In addition, secretin and VIP activate an elevation of cAMP levels, and thus activation of PKA (Wäsle & Edwardson, 2002). On the other hand, a local renin-angiotensin system (RAS) demonstrated to exist in the acinar cell may have a functional role in the regulation of protein secretion by pancreatic acinar cells (Leung & Carlsson, 2001). In this context, angiotensin II was shown to stimulate exocytosis from the pancreatic acinar cells in a dose dependent manner, probably through mediation of intracellular Ca2+ concentration (Tsang et al., 2004). The localization, expression, regulation and function of the RAS in the exocrine pancreas and its potential roles in pancreatic inflammation will be further discussed in Chapters 6 and 10. Figure 2.2 summarizes some important neurohormonal regulators and their respective receptor-mediated signalling pathways that have stimulatory effects on protein secretion from the pancreatic acinar cell.

2.3 Pancreatic Duct Cells The pancreatic duct cells represent a minority of the exocrine portion of the pancreas. Nevertheless, these cells are indispensable for normal functioning of the digestive enzymes secreted by acinar cells and integrity of the duodenal mucosa.

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Pancreatic duct cells are critical because they are responsible for the secretion of the sodium bicarbonate (HCO3 – )-rich alkaline fluid that neutralizes gastric chyme that is emptied into the duodenum. The physiological function of bicarbonate is to provide an optimal pH for pancreatic digestive enzymes and to prevent injury to the duodenal mucosa or peptic ulcer disease. Deficiency of the duct cells thus causes maldigestion and malabsorption. While the acinar cell enzymatic secretion has been studied extensively, the underlying mechanism of ductal secretion is relatively less well understood. Duct cells constitute only 10% of the total number of cells and 5% of the total mass of the human pancreas (Githens, 1988). The duct endings are structurally linked with acinar cells via the centroacinar cells, which have several ductal characteristics and are regarded as the terminals of the ductal tree. Digestive enzymes produced by acinar cells are firstly emptied into the intercalated ducts, followed by intralobular ducts, and finally an interlobular duct. Intralobular ducts merge into the main pancreatic duct, which shares the duodenal opening with the common bile duct at the ampulla of Vater (For the structure and relationship between the duct and acinar cells, please see Fig. 1.1. Chapter 1). There is a sphincter made up of smooth muscle, called the sphincter of Oddi, and its contraction simultaneously regulates bile and pancreatic juices entering into the duodenum. Cells in the intercalated and intralobular ducts are the major producers of HCO3 – . These ducts are lined with principal cells that possess small amounts of rough endoplasmic reticulum, Golgi apparatus and secretory vesicles. Abundant mitochondria are found in the principal cells, reflecting the huge energy demands of the secretory duct cells. The apical membranes of the principal cells possess microvilli while the basal membranes of the principal cells are joined by tight junctions or adherent junctions. Being the largest branches of the network, the interlobular duct cells become columnar in shape and intermingle with goblet cells, which are responsible for mucous production. The pancreatic juice is a clear HCO3 − rich alkaline and isotonic fluid. About 1–2 l of pancreatic juice is produced each day in the adult human body. The concentration of HCO3 − in pancreatic juice is extraordinarily high at 120–140 mmol/l (Domschke et al., 1977), which is fivefold higher than that in plasma. Pancreatic ductal HCO3 − and acinar enzymatic secretion are increased following consumption of a meal. The primary stimulus for HCO3 − secretion by ductal epithelium is regulated by the peptide hormone secretin, secreted by the gut endocrine cells (D-cells), in response to gastric acidic chyme, which enters the duodenum, particularly with a pH below 3.5 (Bayliss & Starling, 1902). Fatty acids and high concentrations of bile salts can also induce the production of secretin by the endocrine D-cells located in duodenal mucosa (Hanssen, 1980). Apart from secretin, multiple stimulatory and inhibitory factors, such as CCK and vagal stimulation, are also involved in the regulation of the pancreatic ductal secretion; this multitude of interacting neurohormonal factors is indicative of the complexity in ductal secretion control (Pandol, 2004). In term of molecular and cellular level, secretion of high concentrations of HCO3 − into the ductal fluid is under the direct control of several ion channels, notably the apical chloride channel called cystic fibrosis transmembrane conductance regulator (CFTR), specifically expressed on the ductal cells. Figure 2.3 presents a schematic

2.3

Pancreatic Duct Cells

19

Fig. 2.3 Regulated secretion of sodium bicarbonate from the pancreatic duct cell. The primary stimulus secretin is released by the duodenal endocrine cells in response to the arrival of gastric chyme. Stimulation of the apical membrane-bound CFTR channel is dependent on the elevation of cAMP levels which, in turn, rests on the activation of secretin receptor located at the basolateral membrane of pancreatic duct cell

of the currently accepted cell model for CFTR-mediated bicarbonate secretion by duct cells. In this model, CO2 enters the cell basolaterally by passive diffusion and carbonic anhydrate, which is expressed within the duct cell, catalyzes the formation of HCO3 − from CO2 and H2 O while concomitantly producing protons or H+ . The protons are transported out of the cell through a Na+ /H+ exchanger on the basolateral membrane. The 2 K+ /3Na+ -ATPase then pumps Na+ out of the cell, thus providing a driving force for the Na+ /H+ exchanger with the establishment of a concentration gradient of Na+ ions. Meanwhile, HCO3 − is secreted out in exchange

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for Cl− by a Cl− /HCO3 − exchanger located in the apical membrane (Melvin et al., 1999). Importantly, the tight coupling of Cl− and HCO3 − secretion depends critically on the regulation and activity of the CFTR channel (Poulsen et al., 1994). Indeed, the CFTR activity or its opening is cAMP dependent which, in turn, is determined by the hormone secretin bound to its receptor located on the basolateral membrane of duct cell. CFTR may be “switched on” for HCO3 − transportation when the luminal Cl− is reduced to about 20 mmol/l (Shcheynikov et al., 2006). The clinical relevance of the CFTR is manifested by a genetic defect of this anion channel in patients with cystic fibrosis (see Section 3.2, Chapter 3). Apart from its established pancreatic secretory function, the pancreatic ductal cell has recently been proposed to be a source of potential progenitors or stem cells. It has been found that the duct cells possess some proliferative capacity for the neogenesis of new ducts, as well as islet cells, even after the morphogenesis of pancreas has been fully completed (Bonner-Weir et al., 2004). An increase in the mitotic activity of the duct cells is obvious when the pancreas is subjected to various degrees of injury, such as duct ligation or streptozotocin-induced destruction of β-cells (Bonner-Weir & Sharma, 2002). Co-expression of several ductal markers and developmental markers, notably PDX1 (a specific pancreatic cell marker), in a subset of duct cells were identified. These findings prompt us to suggest that pancreatic ductal cells may serve as progenitors or stem cells for the generation of new pancreatic cells (Bonner-Weir and Sharma, 2002). Accumulated evidence has further shown that in vitro culture of enriched duct cells from human pancreas or non-obese diabetic (NOD) mice are capable of generating insulin-secreting cells (Bonner-Weir & Sharma, 2002). Moreover, ductal cells have been found to initiate endocrine cell differentiation with the inclusion of several specific transcriptional factors (Heremans et al., 2002). Owing to the lack of specific markers for pancreatic ductal cells, the regenerative capacity of the duct cells remains inconclusive. Further information about pancreatic stem cells, with particular focus on the novel roles of the RAS on their proliferation, differentiation and maturation will be discussed in Chapter 9.

2.4 Pancreatic Islet Cells In contrast with the massive exocrine portion of the pancreas, the endocrine portion represents only 1–2% of the total mass of the organ. Despite their minority, islet cells mediate indispensable functions in glucose homeostasis. The richly vascularized islets receive about 10–15% of the total pancreatic blood flow despite of their small cell population. Amongst the four major types of endocrine cells, β-cells are dominant, constituting about 80% of the total population of islet cells. The four islet cell types are arranged in a well-defined pattern, such that β-cells are located in the centre surrounded by α- and δ-cells in islet periphery. Blood flows from the centre of the islet to the periphery; this unique arrangement provides a paracrine cell–cell interaction within the islets (see Fig. 1.2, Chapter 1). By virtue of this arrangement, α-cells and δ-cells are exposed to high concentrations of insulin so

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as to finely control glucagon and somatostatin release, respectively. β- and δ-cells are distributed throughout the pancreas; α-cells are located exclusively in the tail, body and superior part of the head of the pancreas; F-cells or PP-cells that produce pancreatic polypeptide are present in the middle and inferior parts of pancreas head. The function of pancreatic β-cells is to synthesize and release insulin, which is well known to be an anabolic peptide hormone. It decreases plasma glucose levels by enhancing glucose uptake by peripheral tissues such as adipose and skeletal and suppressing hepatic glucose production. Pre(pro)insulin is cleaved into (pro)insulin during its insertion into the endoplasmic reticulum. (Pro)insulin consists of an amino-terminal β-chain and a carboxy-terminal α-chain as well as a connecting peptide, known as the C-peptide, between the chains. C-peptide enables proper folding of the insulin molecule and formation of disulphide linkages between the α-chain and β-chain. In the endoplasmic reticulum, (pro)insulin is cleaved by a specific endopeptidase known as prohormone convertase into mature insulin. Removal of the C-peptide exposes the end of the insulin chain and thus allows its interaction with insulin receptors (Steiner and Rubenstein, 1997). The free C-peptide and mature insulin are packed into secretory granules in the Golgi apparatus of β-cells and are released into the blood by exocytosis. Only ∼5% of the granules are readily releasable; most of the granules (>95%) are reserved and require further chemical modification to become readily releasable. Thus, only a small proportion of insulin can be released by the β-cells under maximal stimulation.

2.4.1 Beta-Cells Beta-cells are best known for producing insulin in response to changes in plasma levels of major nutrients, particularly glucose, amino acids, and fatty acids. Insulin secretion is also increased by a number of gut hormones such as insulin, glucagonlike peptide-1 (GLP-1), gastric inhibitory peptide (or called glucose-dependent insulinotropic peptide, GIP), secretin and CCK, as well as by vagal and β-adrenergic stimulation. On the other hand, β-cell secretion is decreased by fasting, exercise and somatostatin as well as by enhanced α-adrenergic activity. Beta-cells take up and metabolize glucose, galactose and mannose, and each can provoke insulin secretion by the islet. In this regard, the primary stimulus for insulin release is the nutrient such as amino acid, fatty acids and particularly, glucose. The physiological importance of the primary stimulus glucose can be exemplified by the fundamental concept called, glucose-stimulated insulin secretion (GSIS) (Fig. 2.4). According to this concept, glucose enters the β-cell through a membrane-bound glucose transporter, GLUT2 via facilitative diffusion. The imported glucose is phosphorylated by an enzyme called glucokinase, and a chain reaction of glycolysis ensues, leading to the citric acid cycle in which acetyl-coenzyme A (CoA) and adenosine triphosphate (ATP) are produced. The concomitant increase in the ratio of ATP to ADP inhibits an ATP-sensitive potassium channel so as to reduce K+ efflux, thereby leading to membrane depolarization of the cell. The depolarization activates a voltage-gated Ca2+ channel, which in turn promotes Ca2+ influx through the plasma membrane.

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Fig. 2.4 The basic mechanism of glucose-stimulated insulin secretion (GSIS) by the pancreatic beta-cell. The primary stimulus glucose enters the cell via a facilitative GLUT2 transporter and undergoes glucose metabolism which leads to a rise in ATP/ADP ratio, thus inhibiting ATPsensitive K+ channel. The depolarization of the K+ channel activates a voltage-gated Ca2+ channel which, in turn, triggers a surge of intracellular Ca2+ and finally leads to exocytosis and release of insulin into the blood

A rise in intracellular Ca2+ levels finally triggers exocytosis and release of insulin from the secretory granules into the blood circulation (Schuit et al., 2001). GSIS is well known to exhibit a biphasic action in response to glucose stimulation. Upon glucose challenge, an acute phase of insulin release is evoked, the first-phase insulin secretion, which is characterized by a rapid peak of insulin release and a return to the baseline level. The first-phase of insulin release is followed by a chronic or sustained phase or second-phase insulin secretion, which slowly reaches

2.4

Pancreatic Islet Cells

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a plateau and sustained for the duration of the glucose challenge. The rapid first phase is due to the release of insulin stores of presynthesized insulin while the slow second phase is attributed to the release of newly manufactured insulin or insulin biosynthesis (Maechler et al., 2006). On the other hand, amylin is a peptide produced in beta-cells together with insulin in a ratio of one to one that is thought to regulate gastric motility, renal resorption, and metabolic actions. Formation of islet amyloid deposits from amylin reduces beta-cell mass and insulin secretion, thus acting a key pathophysiological factor in the pathogenesis of diabetes (Wookey et al., 2006).

2.4.2 Alpha-Cells Alpha-cells produce glucagon, which acts in opposition to insulin. In contrast to insulin, its major physiological effect is to increase plasma glucose levels as a catabolic hormone by stimulating de novo synthesis of hepatic glucose via gluconeogenesis. Glucagon secretion is activated by hypoglycemic but inhibited by hyperglycaemic conditions. High plasma levels of amino acids, epinephrine, and vagal activation also stimulate glucagon release. In addition, somatostatin inhibits glucagon release in a paracrine fashion. In general, glucagon can counteract the effect of insulin on glucose homeostasis through its binding to the Gs protein coupled glucagon receptor. Interestingly, it has recently been reported that α-cells can produce a ligand of the growth hormone secretagogue receptor (GHSR), which stimulates the release of growth hormone from the anterior pituitary through a G-protein coupled GHSR (Date et al., 2002). This 28-amino acid peptide called ghrelin was first identified in the stomach (Kojima et al., 1999). Ghrelin stimulates appetite and food intake, while enhancing fat mass deposition and weight gain via its actions on multiple organ systems including the central nervous and gastro-intestinal systems (De Vriese & Delporte, 2008). Recently, it has been proposed that ghrelin-secreting cell called epsilon-cells are identified in the developing and adult human pancreas (Andralojc et al., 2009), indicating that epsilon-cells are ontogenetically and morphogenetically distinct from alpha-cells and beta-cells (see Section 1.2, Chapter 1). In the pancreas, ghrelin has biological effects on islet cell insulin secretion (Date et al., 2002) and acinar enzyme secretion (Zhang et al., 2001) from the endocrine and exocrine pancreas, respectively. In this regard, a locally expressed ghrelin system has been reported in the acinar cells as having a role in the regulation of acinar cell function (Lai et al., 2005). Hence, the acinar cell ghrelin system is subjected to regulation by physiological and pathophysiological stimuli such as gastric acid inhibition, acute pancreatitis, and starvation, suggesting that it is involved in exocrine pancreatic function and dysfunction (Lai et al., 2007).

2.4.3 Delta-Cells and PP-Cells Somatostatin was originally discovered as a neuropeptide produced by the hypothalamic cell, based on its inhibitory action on the release of growth hormone from

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the pituitary gland; it was later found in various cells, including the intestinal endocrine and nerve cells as well as in the pancreas. Apart from its classical actions on the “hypothalamus-pituitary axis”, somatotstain exhibits a wide spectrum of secretory and motor functions on such organs of the gastrointestinal system as the stomach and intestine as well as the exocrine and endocirne pancreas. There are two biologically active forms of somatostatin synthesized from the precursor molecule, preprosomatostatin, called SS-14 and SS-28 consisting of 14 amino acids and 28 amino acids, respectively. In the intestine, SS-14 was identified in both enteric neurons and enteroendocrine cells whereas SS-28 was reported only in endocrine D-cells (Francis et al., 1990). Somatostatin secretion is regulated by nutrients, gastrointestinal hormones, glucagon, and neurotransmitters, and inhibited by insulin. Physiologically speaking, somatostatin is a profound inhibitor of insulin and glucagon secretion as well as a potent inhibitor of acid secretion by the parietal cells of stomach. In general, administration of somatostatin decreases the assimilation rate of all nutrients from the gastrointestinal system (Unger et al., 1978). In the pancreas, somatostatin is produced by the delta-cells of the endocrine islet cells which contributes a relatively few cell population of the islet (about 5%); however, it has profound biological and physiological effects on the endocrine as well as exocrine pancreas. For example, an inhibitory effect of somatostatin on pancreatic exocrine secretion, particularly digestive enzyme release via its mediation of cholecystokinin and secretin, has been previously recognized in humans (Boden et al., 1975, Schlegel et al., 1977). As far as endocrine pancreas is concerned, somatostatin plays profound and inhibitory effects on insulin and glucagon secretion by the islet cells, thus markedly decreasing blood glucose levels in humans and diabetic subjects (Wahren & Felig, 1976). Recently, angiotensing II type 2 receptors (AT2R) have been identified to be co-localized with somatostatin-producing delta-cells in the endocrine pancreas; interestingly, angiotensin II can stimulate a biphasic release of somatostatin in a dose-dependent manner, probably via mediation of the AT2 receptors expressed in δ-cells (Wong & Cheung, 2004). Pancreatic peptide (PP) is produced by a small population of PP-cells or F-cells in the islets. There are only a few F-cells (less than 1% of islet cell population) scattered throughout the islets, primarily concentrated in the dorsal part of the head of the pancreas in a distribution opposite to that of glucagon. PP is a 36-amino acid peptide with a distinctive C-terminal tyrosine amide residue which belongs to the neuropeptide Y and peptide YY family. Its secretion is increased by food ingestion, mainly due to protein and fat content, cholinergic stimulation, and hypoglycaemia, and it is decreased by glucose. Basically, the release of PP is highly dependent upon cholinergic stimulation although some hormonal agents released by meals also regulate postprandial release. Although receptors of PP have been found in gut-brain axis, the physiological function of PP remains obscure. Among the putative roles, PP is known to have diverse effects on the gastrointestinal secretion and motility such as inhibition of exocrine and exocrine pancreatic secretion, stimulation of gastric acid secretion, relaxation of gallbladder motility, and regulation of migrating motor complex of gut smooth muscle (Podolsky, 1994). More recently, it has been reported that elevated plasma levels of PP are associated with increased intra-abdominal fat accumulation and thus insulin resistance in humans (Tong et al., 2007).

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Lai KC, Cheng CHK, Ko WH and Leung PS. Ghrelin system in pancreatic AR42J cells: its ligand stimulation evokes calcium signaling through ghrelin receptors. Int J Biochem Cell Biol 37:887–900, 2005. Lai KC, Cheng CHK and Leung PS. The ghrelin system in acinar cells: localization, expression, and regulation in the exocrine pancreas. Pancreas 35:e1–e8, 2007. Leung PS and Carlsson PO. Tissue renin-angiotensin system: its expression, localization, regulation and potential role in the pancreas. J Mol Endocrinol 26:155–164, 2001. Leung PS and Ip SP. Pancreatic acinar cell: its role in acute pancreatitis. Int J Biochem Cell Biol 38:1024–1030, 2006. Maechler P, Carobbio S and Rubi B. In beta-cells, mitochondria integrate and generate metabolic signals controlling insulin secretion. Int J Biochem Cell Biol 38:696–709, 2006. Melvin JE, Park K, Richardson L, Schultheis PJ and Shull GE. Mouse down-regulated in adenoma (DRA) is an intestinal Cl– /HCO3 – exchanger and is up-regulated in colon of mice lacking the NHE3 Na+ /H+ exchanger. J Biol Chem 274:22855–22861, 1999. Murakami T, Fujita T, Taguchi T, Nonaka Y and Orita K. The blood vascular bed of the human pancreas, with special reference to the insulo-acinar portal system. canning electron microscopy of corrosion casts. Arch Histol Cytol 55:381–395, 1992. Nakagawa A, Stagner JI and Samols E. Suppressive role of the islet-acinar axis in the perfused rat pancreas. Gastroenterology 105:868–875, 1993. Okabayashi Y, Maddux BA, McDonald AR, Logsdon CD, Williams JA and Goldfine ID. Mechanisms of insulin-induced insulin-receptor downregulation. Decrease of receptor biosynthesis and mRNA levels. Diabetes 38:182–187, 1989. Pandol SJ. Neurohumoral control of exocrine pancreatic secretion. Curr Opin Gastroenterol 20:435–438, 2004. Patel R, Singh J, Yago MD, Vilchez JR, Martínez-Victoria E and Mañas M. Effect of insulin on exocrine pancreatic secretion in healthy and diabetic anaesthetised rats. Mol Cell Biochem 261:105–110, 2004. Petersen OH. Human Physiology. Blackwell, Oxford, 2007. Petersen OH. Stimulus-secretion coupling: cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells. J Physiol 448:1–5, 1992. Petersen OH and Ueda N. Pancreatic acinar cells: the role of calcium in stimulus-secretion coupling. J Physiol 254:583–606, 1976. Podolsky DK. Peptide growth factors in the gastrointestinal tract. In LR Johnson (ed), Physiology of the gastrointestinal tract, vol 1, 3rd edn. Raven Press, New York, NY, pp 1–128, 1994. Poulsen JH, Fischer H, Illek B and Machen TE. Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 91:5340–5344, 1994 Saito A, Williams JA and Kanno T. Potentiation of cholecystokinin-induced exocrine secretion by both exogenous and endogenous insulin in isolated and perfused rat pancreata. J Clin Invest 65:777–782, 1980. Scheele G, Adler G and Kern H. Exocytosis occurs at the lateral plasma membrane of the pancreatic acinar cell during supramaximal secretagogue stimulation. Gastroenterology 92:345–353, 1987. Schlegel W, Raptis S, Harvey RF, Oliver JM, Pfeiffer EF. Inhibition of cholecystokininpancreaozymin release by somatostatin. Lancet 2:166–168, 1977. Schuit FC, Huypens P, Heimberg H and Pipeleers DG. Glucose sensing in pancreatic beta-cells: a model for the study of other glucose-regulated cells in gut, pancreas, and hypothalamus. Diabetes 50:1–11, 2001. Shcheynikov N, Wang Y, Park M, Ko SB, Dorwart M, Naruse S, Thomas PJ and Muallem S. Coupling modes and stoichiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6. J Gen Physiol 127:511–524, 2006. Simon T, Marcus A, Royce CL, Chao F, Mendez T and Fink AS. Hyperglycemia alone does not inhibit secretin-induced pancreatic bicarbonate secretion. Pancreas 20:277–281, 2002.

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Steiner DF and Rubenstein AH. Proinsulin C-peptide-biological activity. Science 277:531–532, 1997. Tong J, Utzschneider KM, Carr DB, Zraika S, Udayasankar J, Gerchman F, Knopp RH and Kahn SE. Plasma pancreatic polypeptide levels are associated with differences in body fat distribution in human subjects. Diabetologia 50:439–442, 2007. Tsang SW, Cheng CHK and Leung PS. The role of pancreatic renin-angiotensin system in acinar digestive enzyme secretion and in acute pancreatitis. Regul Pept 119:213–219, 2004. von Schönfeld J, Goebell H and Müller MK. The islet-acinar axis of the pancreas. Int J Pancreatol 16:131–140, 1994. Unger RH, Dobbs RS and Orci L. Insulin, glucagon and somatostatin secretion in the regulation of metabolism. Annu Rev Physiol 40:307–343, 1978. Wahren J and Felig P. Influence of somatostatin on carbohydrate disposal and absorption in diabetes mellitus. Lancet 2:1213–1216, 1976. Wäsle B and Edwardson JM. The regulation of exocytosis in the pancreatic acinar cell. Cell Signal 14:191–197, 2002. Williams JA and Goldfine ID. The insulin-pancreatic acinar axis. Diabetes 34:980–986, 1985. Wookey PJ, Lutz TA and Andrikopoulos S. Amylin in the periphery: an updated mini-review. ScientificWorldJournal 6:1642–1652, 2006. Wong PF and Cheung WT. Immunohistochemical colocalization of type II angiotensin receptors with somatostatin in rat pancreas. Regul Pept 117:195–205, 2004. Zhang W, Chen M, Chen X, Segura BJ and Mulholland MW. Inhibition of pancreatic protein secretion by ghrelin in the rat. J Physiol 537:231–136, 2001.

Chapter 3

Common Pancreatic Disease

3.1 Pancreatitis Pancreatitis is an inflammatory disease of the pancreas characterized by acute and chronic condition as well as varying duration and severity. Acute pancreatitis (AP) is a severe abdominal inflammation, characterized by parenchymal edema, necrosis with occasional presence of pseudocysts, abscess, hemorrhage, and inflammatory cell infiltration (Chan & Leung, 2007a). Its disease spectrum varies from mild edematous to severe disease with fatal complications. Pancreatic acinar cells are very susceptible to inflammation during an episode of AP attack. It is believed that some initiating factors can lead to the premature transformation of inactive pro-proteases into active proteases within the pancreas. The active proteases degrade a number of cellular proteins such as the structural protein F-actin, thus leading to the collapse and malfunction of acinar and pancreatic damage, a process known as autodigestion of the pancreas (Singh et al., 2001).

3.1.1 Etiology and Prognosis Some AP-triggering environmental toxins are known to directly affect pancreatic acinar cells, leading to rupture and necrosis, and eventually an extensive inflammatory response. Epidemiological studies have revealed that the morbidity rate of the disease depends on which regions are examined, ranging from 50 to 800 cases per 1,000,000 people annually. Actually, AP is more prevalent in Caucasian than in Asian populations. The incidence rate is 700–800 and 150–420 per million annually in the USA and UK, respectively (Banks, 2002); however, it is just 106– 205 per million annually in Japan (Sekimoto et al., 2006). Similar studies in China and Hong Kong have been inconsistent. In 2007, there were only 1976 patients with AP admitted to hospital from 15 Chinese tertiary care centres during the period from 1990 to 2005 (Bai et al., 2007). Despite this discrepancy, there are an increasing number of individuals with AP, probably due to life-style changes and increased exposure to risk factors in recent decades. P.S. Leung, The Renin-Angiotensin System: Current Research Progress in The Pancreas, Advances in Experimental Medicine and Biology 690, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9060-7_3, 

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The diagnosis of AP entails several characteristic clinical features, such as abdominal pain at the upper epigastric region, vomiting, and elevation of plasma pancreatic enzyme levels, such as α-amylase and lipase. Hyperstimulation or pancreatic lesions might account for leakage of pancreatic enzymes into circulation. It should be emphasized that elevated levels of plasma pancreatic enzymes are a commonly used indicator for AP, though they do not generally reflect the degree of disease severity. It is noted that the elevation of plasma α-amylase tends to be cleared most rapidly from the circulation and it is the first enzyme to fall during resolution of AP. In view of this fact, if a patient with AP presents more than a few days after onset of abdominal pain, serum amylase can be normal or only slightly elevated. In this case, serum lipase is however cleared less rapidly and thus this enzyme is more likely to remain elevated in patients whose onset of symptoms is several days earlier. In addition, serum amylase levels can also be elevated in non-AP conditions such as pancreatic duct obstruction, pancreatic cancer, appendicitis, bowel obstruction and renal failure. On the other hand, measurement of serum C-reactive peptide and interleukin-6 (IL-6) provides additional information, particularly within 48 h of the onset of AP symptoms (Davies & Hagen 1997). For further evaluations, approaches such as ultrasonography and computed tomography imaging are more effective and reliable; these diagnostic procedures offer a better understanding of the cause of the disease, including the presence of gallstones or dilatation of the common bile duct. In the clinical setting, about 80% of AP can be etiologically determined while the remaining cases are classified as idiopathic or without a known cause. Alcoholism and gallstone obstruction (choledocholithiasis) are the two major causes of the disease, accounting for about 35 and 45% of AP cases, respectively. Other causes of AP, to name but a few, include drug uses, trauma, genetic and infection. The cardinal feature of alcoholism-induced AP is characterized by the formation of proteinaceous plugs in bile ducts thus activating proteolytic enzymes and leading to pancreatic autodigestion. Gallstone obstruction-induced AP is, however, due mainly to gallstone formation which results in blockage of ampulla of Vater and thus reflux of biliary and pancreatic juices into pancreatic ducts, eventually leading to pancreatic inflammation. Different countries and regions have various proportions of pancreatitis caused by alcoholism and gallstones. Alcoholism is a major cause in some regions while gallstones may be a major cause in others. A similar incidence rate between these two etiological factors has also been reported (Gullo et al., 2002). However, it should be noted that the risk of acute alcoholic pancreatitis and acute gallstone-induced pancreatitis is around 2–3% and 0.63–1.48%, respectively, suggesting that these two risk factors can not fully account for the incidence of AP. In fact, idiopathic pancreatitis accounts for the third most common cause of AP clinically. Other risk factors including hyperlipidemia, viral infection (HIV, mumps and hepatitis B), and endoscopic retrograde cholangiopancreatiography (ERCP), as well as surgical procedures and medications may be implicated in the development of AP (Chan & Leung, 2007a). The AP recurrence rate is relatively high. A cohort study in Sweden reported that about 21% of AP patients suffer a recurrence, with nearly 70% of them

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Pancreatitis

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exhibiting a second episode within three months of the first episode (Andersson et al., 2004). A similar follow-up study conducted in Japan indicated an overall recurrence rate of 37% (Sekimoto et al., 2006). If choledocholithiasis is not treated properly, the recurrence rate of gallstone-induced pancreatitis could reach 32–61% (Delorio et al., 1995). Patients with alcoholism-induced pancreatitis and idiopathic pancreatitis have been reported to have relapse rates of around 46 and 3.2%, respectively, indicating that recurrence varies in an etiology-dependent manner (Pelli et al., 2000). Some patients may gradually develop chronic pancreatitis (CP) if recurrent AP is not well controlled. Basic research studies have revealed that repeated episodes of AP result in a progressive development to CP. The so-called “necrosis-fibrosis theory” hypothesizes that residual pancreatic damage, especially necrosis, can gradually lead to parenchymal destruction and fibrosis replacement (Ammann & Muellhaupt, 1994). It is believed that repeated acinar injury leads to activation of pancreatic stellate cells (PSCs). The activated PSCs secrete fibrogenic factors, thus triggering progressive substitution of functional exocrine pancreas and subsequent development of CP (Leung & Chan, 2009). It should be emphasized that AP and CP are two distinct diseases with discrete features in terms of pancreatic morphology and clinical outcome. Patients with AP usually exhibit pancreatic swelling (edematous or necrotic cell death), while the pancreata of CP patients usually undergoes atrophy and apoptosis. In AP, the damage is confined to the exocrine pancreas and exocrine dysfunction leads to pancreatic exocrine insufficiency; meanwhile, CP or repeated episode of AP attack causes endocrine dysfunction that emerges from advanced stage of CP, notably in the form of pancreatic fibrosis which accounts for not only pancreatic exocrine but also endocrine insufficiency, the latter being attributable to impaired glucose tolerance or diabetes. Mortality is the major complication of severe AP. The overall mortality rate of AP lies between 7.5 and 20%, depending on the diagnostic criteria chosen and the age of the patients. Most patients are susceptible to death within the first few weeks after the onset of symptoms. Early mortality is associated with the development of systemic inflammatory response syndrome (SIRS), aggravated by multiple organ dysfunction syndrome (MODS). In this context, death is generally due to local complications, notably occurrence of infection resulting from pancreatic necrosis. The clinical parameters of MODS include dyspnea, shock, bleeding tendency, and elevated blood urea (Sekimoto et al., 2006). In terms of organ failure, pulmonary dysfunction is commonly observed. Acute respiratory distress syndrome (ARDS) is usually the primary manifestation of AP-associated complications and is believed to trigger MODS (Bosma & Lewis, 2007). On the other hand, the development of ARDS may further exacerbate the severity of pancreatitis since hypoxia is a well-known enhancer of inflammatory responses. Undoubtedly, the more organs that fail, particularly if the liver and kidney are involved, the higher the mortality rate is (McFadden, 1991). The mortality rate in AP-induced ARDS surges when renal failure has ensued. Actually, there is a close association between pancreatic necrosis and development of SIRS as well as MODS. It is believed that necrotizing pancreatitis causes the release of overwhelming pro-inflammatory mediators and,

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3 Common Pancreatic Disease

subsequently, over-activates inflammatory responses, leading to SIRS and distant organ lesions and thus death ensues.

3.1.2 Treatments Although AP has been studied for decades, promising and effective therapy is still not available. In most cases, patients develop mild, self-limiting AP, requiring no special treatment or surgery. Traditional approaches are generally palliative and nonspecific including analgesic (opiate) administration for pain relief, intravenous fluids for volume depletion and nothing by mouth for pancreatic rest. By doing so, the patients may be discharged from the hospital within weeks. However, around 20% of patients develop severe AP, concomitant with lethal complications that bring the mortality rate up to 20–25% in these patients, compared with 1–3% in a mild attack. In such cases, patients should be monitored under intensive care unit and surgery should be justified in such case as gallstone-induced AP. Enteral/parentenal nutrition, antibiotic treatment, surgical removal of necrotic tissue, and some surgical manipulations such as cholecystectomy are the first-line treatment modalities for clinical AP (Makola et al., 2007). However, many such treatments are rather passive in nature (e.g. enteral/parentenal nutrition) while others are invasive (e.g. surgical management). Most of these treatments are targeted for the complications of the disease (e.g. sepsis or pain management) rather than the primary insults. In view of this fact, clinicians are in search of new effective alternatives for curing AP. Potential therapeutic approaches derived from basic research include, but are not limited to, the protease inhibitor gabexate, the antisecretagogue agent octreotide, the anti-inflammatory drug lexipafant, and an antioxidant regimen. Unfortunately, thus far, these potential therapeutic methods have yielded unsatisfactory or marginal results and warrant further extensive investigation (Chan & Leung, 2007a; Leung & Chan, 2009). Emerging data from basic research have shed some lights on potential therapeutic strategies against AP, such as cyclooxygenase (COX)-2 inhibition, substance P antagonism, and heat shock protein (HSP) activation. COX-2 expression has been found to be up-regulated in caerulein-induced pancreatitis in both mice (Song et al., 2002) and rats (Zhou et al., 2004). Treatment with specific COX-2 inhibitors, such as celecoxib and NS-398, could alleviate pancreatic injury (Song et al., 2002). Genetic knock-out of COX-2 leads to resistance against experimental pancreatic injury and its associated lung injury (Song et al., 2002). In addition, the expression of substance P, the physiological agonist of neurokinin 1 receptor (NK1R), has been shown to be upregulated during experimental pancreatitis, thus implicating its role in AP-induced lung injury (Bhatia et al., 1998; Lau et al., 2005a); a pharmacological antagonist for its receptor was also protective against caerulein-induced pancreatitis and associated lung damage (Lau et al., 2005a). On the other hand, the chaperon protein and HSP have also been closely linked with AP pathogenesis; secreatagogue and arginine-induced pancreatitis was shown to provoke pancreatic HSP protein expression and HSP induction was shown to be protective against experimental

3.2

Cystic Fibrosis

33

reorganization of cytoskeleton proteins (protective)

ROS ↑

Heat Shock Protein↑

Angiotensin II ↑

??? Phospholipase A2 ↑

Acute Pancreatitis

Substance P ↑ Vascular permeability ↑???

Chemotaxis ↑???

Cyclooxygenase-2 ↑

NFκB activation

prostaglandin ↑

Fig. 3.1 Schematic diagram illustrating the simplified relationship between AP pathophysiology and some novel biological factors (modified from Chan & Leung, 2007a)

pancreatitis (Tashiro et al., 2001). Last but not least, we have shown that angiotensin II, the vasoactive peptide, is crucial in mediating pro-inflammatory responses during AP (Chan & Leung, 2007b). The novel roles of angiotensin II and its type 1 receptor (AT1R) blockade and thus its clinical relevance to pancreatic inflammation will be further discussed in Chapter 10. Potential pathogenetic factors of acute pancreatitis implicated and discussed above in the pathogenesis of AP are summarized in Fig. 3.1.

3.2 Cystic Fibrosis Cystic fibrosis (CF) is an autosomal recessive disease commonly found about once in every 25,000 births in the Caucasian population while it occurs rare in native Africans and Asians. The disease is a result of a genetic defect leading to abnormal sodium chloride and water movement across the epithelial membranes. Where such membranes line the pancreatic duct and small respiratory airways, they produce dense and viscous mucus that cannot be properly cleared, thus causing pancreatic insufficiency and pulmonary disorder. The genetic defect involves a particular membrane protein expressed in the epithelial cells, called cystic fibrosis transmembrane conductance regulator (CFTR). Mutations in the CFTR are categorized into five groups according to their repercussion on CFTR protein synthesis and its chloride channel function. Class I or stop codon mutations result in truncated non-functional CFTR; Class II mutations consist of aberrantly folded CFTR protein that is degraded by the cell quality control system; Class III mutations lead to defective regulation of the CFTR protein; Class IV mutations cause defective chloride conductance; and

34

3 Common Pancreatic Disease

Class V mutations interfere with normal transcription, thereby reducing the amount of normal CFTR (Proesmans et al., 2008).

3.2.1 Etiology and Prognosis One of the genetic mutations of CF gene is due to a single mutation (amino acid 508) located on the long arm of chromosome 7 that codes for the CFTR protein. This genetic defect is characterized by abnormalities in exocrine gland function that result in altered ion composition and increased viscosity of epithelial cell secretions. The affected tissues include the secretory cells of the sweat glands, salivary glands, small intestine, lungs, vas deferens, and the exocrine pancreas (Voynow & Scanlin, 2005). CF is manifested by elevated electrolyte concentrations, reduced ion permeability, and impaired luminal ion secretion upon stimulation by secretagogues (Tucker et al., 2003). Thus, the defect in perturbed salt and water transport leads to secondary alterations of pancreatic, intestinal and pulmonary functions. Pancreatic exocrine insufficiency is clinically obvious in 85–90% of CF patients (Baker et al., 2005). As mentioned, CFTR is expressed in various cell types, including the pancreas. It is comprised of two membrane-spanning domains and two nucleotide-binding domains separated by a regulatory R domain. The two membrane-spanning domains form a low-conductance chloride channel pore. It is regulated by ATP binding and hydrolysis at the nucleotide-binding domains following initial phosphorylation of the R domain. CFTR functions as a chloride channel in the apical membrane of epithelial cells that regulate ion transport (Schwiebert et al., 1999). It has inhibitory effects on apical Na+ permeability across epithelia while activating non-CFTR chloride channels. Owing to the widespread presence of CFTR throughout the body, CF is a multisystem disorder affecting many organs, especially the gastrointestinal tract, pancreas and lungs. Failure of the function of CFTR results in impaired chloride transport at the apical surface of epithelial cells and dysregulation of other transporters, such as the chloride-coupled bicarbonate transport and sodium channel activity (Reddy et al., 1999; Choi et al., 2001). The aqueous medium of the intestinal lumen becomes hyper-viscous in CP patients due to excessive and abnormal intestinal mucoprotein (Forstner et al., 1984). In the pancreas, these viscous secretions cause luminal obstruction of pancreatic ducts, leading to acinar cell destruction, fibrosis, and ultimately pancreatic exocrine insufficiency which is characterized by a decrease or absence of digestive enzymes and concomitant with maldigestion and malabsorption of nutrients. In view of the close interaction between exocrine and endocrine pancreas, patients with CF usually develop with pancreatic exocrine along with endocrine insufficiency as manifested by other pancreatic conditions such as acute and chronic AP, pancreatic surgery and pancreatic cancer (Czako et al., 2009). The pathophysiology of lung disease in CF is more complex than that of pancreatic disease. A major finding is that the airway mucus is thick and viscous as a result of insufficient fluid secretion into the airway. The lung epithelium secretes fluid in

3.2

Cystic Fibrosis

35

a mechanism that requires CFTR while absorbing fluid that requires apical ENaC sodium channels. In patients with CF, the reduced activity of CFTR shifts the balance more toward absorption, and a thick mucous layer is generated that inhibits the ciliary clearance of foreign bodies. The result is an increased rate and severity of infections and thus inflammatory processes that contributes to the destructive process in the lung (Ingbar et al., 2009). In contrast, the levels of salivary secretion are normal or raised while lingual lipase levels are elevated (Guy-Crotte et al., 1996). Although CF is the most common lethal genetic disease in Caucasians, there is often a delay between the onset of symptoms and definitive diagnosis. Blood spot CF screening tests can be performed in newborn babies. A definitive diagnostic sweat test will then be given if the screening suggests CF. About one in five people with CF are diagnosed at birth when their gut is blocked by extra thick meconium.

3.2.2 Treatments The conventional treatment of CF is usually palliative, alleviating signs and symptoms and treating organ dysfunction, including replacement of pancreatic enzymes, vitamin and nutritional supplements, airway clearance techniques, daily physiotherapy and antibiotics for pulmonary infections (Davis et al., 1996; Ramsey, 1996). Despite advances in treatment, there remains no cure for CF. It has become apparent that there is a need for a more effective and convenient therapy. New therapies directed at the basic defect represent the only potential approach to truly treating CF. The identification of the gene responsible for CF (CFTR protein), in 1989, provided insight into a potential treatment for CF. Gene therapy, the transfer of a normal copy of the CFTR gene into the lungs of CF patients, has been proposed as an attractive alternative to the conventional approach (Riordan, 2008; Ratjen, 2007). Genes are most commonly transferred into cells through viruses and liposomes. Viruses have evolved to enter the cells of the body efficiently. Scientists have harnessed this property for gene therapy by inserting a copy of the therapeutic gene of interest into the virus, which then directs it into the cell. The adenovirus is an example of a virus that has been used extensively for gene therapy. On the other hand, liposomes are fatty substances that naturally adhere to the surface of cells, thus facilitating entry into cells. The CF gene could potentially be transferred into the airway cells; however, considerable challenges lie ahead, particularly with regard to efficiency of gene transfer and persistence of transferred gene expression (Atkinson, 2008). The field is moving forward rapidly, particularly pertaining to the development of better virus and liposome vectors. Apart from the aforementioned state-of-the-art methodology, there are a number of potential drugs which are emerging for the treatment of CF and are in clinical trials. They include some anti-bacterial formulations, anti-inflammatory agents, ion channel modulating agents, and agents that correct the underlying gene defect (Jones & Helm, 2009). The pathogenetic pathways and common symptoms of CF are briefly summarized in Fig. 3.2.

36

3 Common Pancreatic Disease

Fig. 3.2 The pathogenesis of cystic fibrosis disease and its common signs and symptoms

CFTR mutation CFTR protein Ion transport Altered secretions Blocked ducts Impaired mucosal defense Infection & Inflammation

Cystic Fibrosis

Symptoms • Salty sweat • Intestinal block • Fibrotic pancreas • Failure to thrive

• Recurrent bacterial lung infections • Congenital bilateral absence of the vas deferens (CBAVD) • Filled sinuses

3.3 Pancreatic Cancer 3.3.1 Etiology and Prognosis Pancreatic cancer is a deadly malignant disease with an extremely high mortality rate in humans. It is classified into tumours arising from the pancreatic exocrine tissue (ductal adenocarcinoma) and pancreatic endocrine tissue (insulinoma, glucagonoma, gastrinoma and VIPoma), as well as atypical neoplasms (lymphomas, mesotheliomas and sarcomas) (Hruban & Adsay, 2009). It is the fourth leading cause of cancer in the United States, with an estimated 37,680 new cases and 34,290 deaths in 2008 (Jemal et al., 2008). Ductal adenocarcinoma accounts for approximately 80% of all pancreatic cancers (Lewis, 2006). Only 23% of patients with pancreatic cancer of exocrine origin will survive for 1 year, while about 4% will survive for 5 years. In contrast, pancreatic endocrine tumours are relatively rare accounting for less than 2% of pancreatic neoplasms. In this section, we thus focus on the ductal adenocarcinoma as an example of pancreatic cancer for further discussion below. The risk of developing pancreatic cancer is much higher beyond the age over 50 and is more prevalent in males. Risk factors include family history, cigarette smoking, a diet rich in fat and meat, and exposure to chemicals. In addition, patients

3.3

Pancreatic Cancer

37

with diabetes mellitus, chronic pancreatitis and gastric surgery are more susceptible to the disease (Dunphy, 2008). Diagnosis of pancreatic cancer in an early stage is extremely difficult, mainly due to the nonspecific symptoms and the lack of accurate diagnostic tests. Hence, more than 80% of patients with pancreatic cancer are diagnosed only in advanced or late tumour stages (stage II–IV), often when the tumours metastasize without a recourse of tumour resection (Canto, 2008). Furthermore, the propensity for tumour metastasis and intrinsic resistance to conventional chemoradiation treatments renders pancreatic cancer very difficult to handle clinically. Surgical resection of localized tumours is the only potentially curative option available for pancreatic cancer patients (Ozawa et al., 2001). Unfortunately, pancreatic cancer is a highly aggressive cancer and, with the late diagnosis that characterizes many cases, only about 10–15% of patients are considered suitable for the surgical option. There are two standard resection procedures, including resection of the pancreatic head and of the left-side pancreatic tissues. In the case of a local advanced tumour, a total pancreatectomy may be chosen for particular patients (Loos et al., 2008a). Pancreatic surgery has significantly improved and the associated postoperative morbidity and mortality have decreased markedly over the last three decades. Despite these advances, the prognosis for patients with pancreatic cancer remains poor and this poor prognosis is the reason for pancreatic cancer’s high mortality rate.

3.3.2 Treatments Complete surgical resection of the tumour is only possible in 10–25% of patients with pancreatic cancer, while about half of them are subjected to adjuvant therapies. Chemotherapy with 5-fluorouracil (5-FU), cisplatin, oxaliplatin, mitomycin C, and doxorubicin in mono- or combination treatments are the major options for pancreatic cancer. In the case of locally unresectable or metastatic adenocarcinoma, chemotherapy was found to enhance overall 1-year survival, but not to appreciable improve long-term (5-year) survival (Loos et al., 2008b). In 1996, gemcitabine was approved as the standard chemotherapeutic agent in monotherapy or combination therapy for locally advanced and metastatic pancreatic cancer (Barton-Burke, 1999; Hui & Reitz, 1997). Gemcitabine has been reported to improve survival and alleviate disease-related symptoms (Almhanna & Kim, 2008). Single chemotherapy with gemcitabine is considered the standard for patients with advanced pancreatic cancer along with other drugs including 5-FU. Meanwhile, targeted therapies inhibiting specific pathways for the growth and progression of malignant tumors are under intensive investigations. The epidermal growth factor receptor (EGFR) mediated pathway is one of the most promising targets for the treatment. In over 90% of cases, EGFR is over-expressed to stimulate pancreatic tumour growth (Lemoine et al., 1992). Indeed, the EGFR tyrosine inhibitor erlotinib has been shown to increase median survival and, in 2005, the U.S. FDA approved the use of erlotinib in combination with gemcitabine for treating locally advanced, unresectable, or metastatic pancreatic cancer (Danovi et al., 2008). Gene therapy using antisense oligonucleotides or ribozymes could also correct a genetic defect or mutation. For example, K-ras and p53 tumour suppressor gene mutations are found in about 70%

38

3 Common Pancreatic Disease

of pancreatic cancer and have been predominant targets in preclinical gene therapy studies (Danovi et al., 2008). Despite these recent advances, treatment of pancreatic cancer remains difficult and relatively ineffective due to its intrinsic broad resistance to cytotoxic drugs. Some recent studies have focused on the transcription factor nuclear factor kappa B (NF-κB), which is proposed to be a central determinant in the induction and manifestation of chemo-resistance in pancreatic cancer cells. Since constitutive activation of NF-κB is a hallmark of pancreatic cancer accounting for profound chemoresistance, suppression of NF-κB activity might be a useful strategy for increasing sensitivity towards cytostatic drug treatment (Sebens et al., 2008). However, solid tumors apparently exhibit comprehensive protection from induction of apoptosis, so pharmacological inhibition of NF-κB only appears to be insufficient. Thus, NF-κB inhibition should be used as a chemo-sensitizing adjuvant in combination with other cytostatic drugs. Several new immunotherapies have been developed for potential treatment of pancreatic cancer. Chemotherapy, which reduces tumour cells by preferentially poisoning proliferating cells, is not a cancer cure per se. Immunotherapy, however, could potentially eliminate quiescent tumour (stem) cells. Although immunotherapy is in the early stage of development and has not yet replaced conventional chemotherapy, it may in the future be an additional approach in fighting cancer and eradicating tumour stem cells (Schulenburg et al., 2006). Table 3.1 summarizes the different forms of pancreatic tumours that arise from exocrine and endocrine origins, together with their characteristics and the current treatments.

3.3.3 Alternative Approach Using Traditional Chinese Medicine Based on herbal medicine knowledge that has accrued over centuries, traditional Chinese medicine provides a potential approach for alternative treatments to pancreatic cancer. Chinese herbal extracts have been extensively investigated in various clinical trials of anti-tumour studies, including studies of leukemia, lung, ovarian, breast and pancreatic cancers. Indeed, a number of Chinese herbs have been identified as having anti-cancer properties (Wang et al., 2005). For example, Curcumin has been reported to possess a wide range of beneficial properties, including anti-inflammatory, antioxidant, chemopreventive and chemo-therapeutic activities. Curcumin influences multiple signalling pathways, including the following: cell survival pathways regulated by NF-κB, Akt, and growth factors; Nrf2-dependent cytoprotective pathways; and matrix metallo-proteinase-dependent metastatic and angiogenic pathways (Park et al., 2005; Zheng & Chen, 2004). Additionally, the traditional Chinese medicinal Brucea javanica was recently shown to exhibit various biological activities such as anti-malarial (O’Neill et al., 1987), anti-inflammatory (Hall et al., 1983), and hypoglycaemic effects (NoorShahida et al., 2009), thus implicating its clinical use for the treatment of various diseases. Brucea javanica is a shrub of about 3-meter height. The fruit of Brucea javanica, or Ya Dan Zi in Chinese (meaning crow bile fruit), contains oils (glyceroltrileate,

Pancreatic endocrine tumor

Extremely rare and is not aggressive as adenocarcinomas. It has unusually large cells Rare but very distinctive tumor. It grows along the pancreatic duct and appears to be a fingerlike projection into the duct

Giant cell tumor

Insulinoma

Pancreatoblastoma

Intraductal papillarymucinous neoplasm (IPMN) Mucinous cystadenocarcinoma

Adenosquamous carcinoma

Rare, cystic, fluid-containing pancreas tumor and can develop into cancer over time. The space within the spongy tumor is filled with a think fluid called mucin Rare malignant tumor occurs primarily in children, and called pancreatic cancer of infancy Produce large amounts of insulin which result in hypoglycemia

The most common type of pancreatic cancer, accounting for 75% of all pancreas cancer; nearly all of these are ductal adenocarcinoma; cause back pain when tumor grow large and invade nerves Rare cancerous tumor produces excessive amounts of digestive enzymes. Unusual skin rashes, joint pain and increased increased eosinophils level Similar to adenocarcinoma that it forms glands, but it flattens as it grows. It can mimic other types of cancer that show squamous differentiation

Adenocarcinoma

Pancreatic exocrine tumors

Acinar cell carcinoma

Characteristics

Types

Origin

Surgical resection Chemotherapy: Streptozocin, dacarbazine, doxorubicin and 5-Fu Combination therapy

Radiation therapy and chemoradiotherapy

Second line chemotherapy: 5-flourouracil (5-Fu), irinotecan, celecoxib, cisplatin and oxaliplatin Combined therapy: ICM-C225 + Gem, erlotinib + Gem

Surgical resection: pancreaticoduodenectom, total pancreatectomy and distal pancreatectomy First-line chemotherapy: Gemcitabine

Treatments

Table 3.1 A brief summary of classification, characteristics and current treatments of exocrine and endocrine tumors of the pancreas

3.3 Pancreatic Cancer 39

Origin

Characteristics

Produce excessive amounts of glucagon which result in severe dermatitis, mild diabetes, stomatitis, anemia, and weight loss

Release large quantities of the hormone gastrin into the blood stream leading to severe duodenal ulcers and persistent diarrhea

Releasing large amounts of the hormone VIP into the blood stream. Symptoms include watery diarrhea, hypokalemia, and either achlorhydria or hypochlorhydria.

Less common, releasing large quantities of the hormone somatostatin into the blood stream

Types

Glucagonoma

Gastrinoma

VIPoma

Somatostatinoma

Table 3.1 (continued)

Distal pancreatectomy Standard chemotherapy: streptozocin and dacarbazine Octreotide: Reduce elevated glucagon levels, and control the hyperglycemia and dermatitis Surgical resection Chemotherapy: Proton pump inhibitors, like lansoprazole, pantoprazole esomeprasole, in high doses to control hypersecretion of gastric acid Surgical excision Chemotherapy: Octreotide to reduce circulating VIP levels and control diarrhea No specific chemotherapy for VIPoma patients Combination treatment with intravenous 5-FU and streptozotocin or doxorubicin and 5-FU

Treatments

40 3 Common Pancreatic Disease

3.3

Pancreatic Cancer

41

Fig. 3.3 Photographs of Brucea javanica. (a) The plant. (b) The fruit. (c) The dry fruit. (d) Chemical structure of brucein D

oleic acid, linoleic acid), alkaloids, quassinoids, bursatol, brucein A, B, C, D, E, F and H etc (Fig. 3.3). In term of cancer, extracts or compounds from Brucea javanica, particularly brucein D, have anti-proliferative and cytotoxic effects on a number of cancer cell types; they include, but are not limited to, the lung, liver, breast and oesophageal cancers (Lau et al., 2005b). In our laboratory, we have recently performed preliminary screening tests on nine commonly used Chinese herbal medicines for anti-pancreatic cancer activities. Among these, an ethanolic extract of Brucea javanica fruit has also been identified to possess potent cytotoxicity that induced marked apoptosis in several human pancreatic cancer cell lines, including PANC-1, SW1990 and CAPAN-1 while exhibiting less cytotoxic action on Hs68 cells (a human foreskin fibroblast cell line); specifically, it produced chromatin condensation and fragmentation as well as activation of the

42

3 Common Pancreatic Disease

BD

ROS i.e. superoxide IkB-α p50 NF-kB

p65

p-38 MAPK

P

IkB-α

degradation Caspase 8 IkB-α

p50

Bcl-2

p65

Bak

P

Cytochrom c

Bcl-2 XIAP

Caspase 9

p50 p65

P

kB site

Caspase 3

DNA fragmentation

Apoptosis

Cell survival

Fig. 3.4 A schematic presentation of the proposed mechanism (s) by which brucein D induces apoptosis and anti-apoptosis in PANC-1 cells

proteolytic cleavage of caspase 3 in these human pancreatic cancer cell lines (Lau et al., 2008). Furthermore, brucein D, a quassinoid compound found abundantly in Brucea javanica fruit, displayed potent anti-proliferative activity and induced apoptosis in PANC-1 cells through activation of the p-38 mitogen-activated protein kinase (MAPK) signal transduction pathway (Lau et al., 2009). Both intrinsic and

3.4

Diabetes Mellitus

43

extrinsic apoptotic pathways are activated following p38-MAPK phosphorylation and, ultimately, the effector caspase 3 is activated, causing irreversible apoptotic cell death in pancreatic cancer cells. On the other hand, Brucein D treatment depletes intracellular glutathione levels in PANC-1 cells, while triggering activation of the NADPH oxidase isoforms p22phox and p67phox , and increasing superoxide generation. Although intracellular reactive oxygen species and NADPH oxidase activities can be inhibited by the presence of an antioxidant (e.g. N-acetyl cysteine), inhibition of NF-κB activity in brucein D-treated cells appears to be independent of ROS changes (Lau et al., 2010). These results suggest that brucein D elicits apoptosis in pancreatic cancer cells by activating the redox-regulated p38 MAPK signalling pathway and reducing anti-apoptotic activity through inhibition of NF-κB activation. Taken the above substantial data together, these findings provide experimental evidence that supports the potential use of traditional Chinese medicinal such as Brucea javanica fruit extract, in the treatment of pancreatic cancer and, more importantly, brucein D is a promising candidate compound for further development into a mechanism-driven anti-pancreatic cancer agent. A schematic presentation of the proposed mechanism(s) by which brucein D induces MAPKmediated apoptosis and NF-κB-mediated survival in PANC-1 cells is presented in Fig. 3.4.

3.4 Diabetes Mellitus 3.4.1 Etiology and Prognosis Blood glucose concentration has a normal range of 3.8–7 mmol/l (70–125 mg/dl). When the body is not able to maintain normal glucose status, glucose cannot enter into cells to supply them with energy. Prolonged high blood glucose levels, a state known as hyperglycemia, ultimately leads to diabetes mellitus. The American Diabetes Association (ADA, 2009) distinguishes among normoglycemia, impaired glucose tolerance (IGT, also known as pre-diabetes), and overt diabetes mellitus based on fasting plasma glucose (FPG) levels and 2-h oral glucose tolerance test (OGTT) results as follows: normoglycemia is FPG angiotensin (1–7) ≫ angiotensin (1–6)] (Chappell et al., 1992, 1995). Using 125 I-[Sar1 , Thre8 ]angiotensin II, studies of distribution sites have shown a high density of angiotensin II binding sites in pancreatic acinar cells. Interestingly, the most predominant binding sites for angiotensin II in AR42J cells are AT2 receptors, with a molecular weight of approximately 110 kDa, which is greater than that based on the analysis of AT2 receptor protein sequence. Specific antagonism of the AT2 receptor, but not the AT1 receptor, abolished angiotensin II binding activities (Chappell et al., 1995, 2001).

6.1

Acinar Cell RAS

91

Some angiotensin II binding sites on AR42J cells were also sensitive to AT1 receptor antagonism, however, indicating that there are AT1 receptors on the cells. There are two isoforms of AT1 receptor, namely AT1a and AT1b receptor subtypes that are expressed in AR42J cells but the AT1a receptor subtype was found to be the predominant isoform on AR42J cells, as evidenced by RT-PCR analysis (Leung & Chappell, 2003). It has been shown that AT1 receptors generally mediate angiotensin II-induced changes in intracellular Ca2+ levels by activation of phospholipase C, through a pertussis toxin-insensitive pathway as well as stimulating inositol 1,4,5-trisphosphate (IP3 ) and influencing cellular Ca2+ stores, thus finally eliciting biological action of angiotensin II (Yin et al., 2003). In consistent with our expression data, AT1a receptor has also been confirmed to be the predominant subtype of AT1 receptor expressed in the AR42J cells (Cheung et al., 1999). More importantly, a functional AT1a receptor has been characterized in AR42J cells and angiotensin II were found to mediate AT1 receptor activation and thus produce a dose-dependent increase in α-amylase release and inositol phosphate production from the cell line; indeed, high-performance liquid chromatography analysis showed that angiotensin II stimulates the rapid accumulation of IP3 (Cheung et al., 1999). In this study, it is intriguing that angiotensin II-induced α-amylase secretion could be blocked by losartan, a specific antagonist for AT1 receptor while CGP42112, a selective agonist for AT2 receptor, did not exhibit an appreciable effect on amylase release at micromolar concentrations (Cheung et al., 1999). In addition to angiotensin II receptors, renin, angiotensinogen and ACE along with angiotensin II, angiotensin III and angiotensin (1–7), were expressed in the pancreatic acinar AR42J cell line, as demonstrated by RT-PCR analysis and radio-immunoassay coupled with highperformance liquid chromatography (Leung & Chappell, 2003). Table 6.1 provides a summary of the key RAS components that are expressed in the rat AR42J cell line as well as acinar cells from different animal species. Apart from the pancreatic rat acinar cell line, expression of acinar RAS components has also been detected in the whole pancreas and isolated acinar cells

Table 6.1 The expression of RAS components in acinar cell line and acinar cells from various animal species. “+” and “–” denote the presence and absence of the respective component, respectively RAS components/cell types Rat AR42J cell Canine acinar cell Rat acinar cell Mouse acinar cell Human acinar cell

Renin ACE Angiotensinogen Angiotensin II AT1 receptor AT2 receptor + –

+ –

+ +

+ +

+ +

+ +

– –

+ –

+ –

+ +

+ –

+ –

–

–

+

–

+

–

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6 Pancreatic RAS

from various animal species. Angiotensinogen mRNA and protein, angiotensin II, angiotensin III, angiotensin (1–7), and high affinity binding sites for angiotensin II were found to be present in dog pancreas (Chappell et al., 1991). Amongst the angiotensin peptides, angiotensin II is the most abundant bioactive peptide, with concentrations present at approximately fivefold those of angiotensin (1–7). Intriguingly, the concentrations of these three angiotensin peptides in the pancreatic tissue levels are three-fold higher than those present in the blood circulation. Specific binding sites for 125 I-angiotensin II were observed overlying the pancreatic acinar cells, and arteriolar and duct tissues, whereas angiotensin I and renin-like activity were not detected in the exocrine pancreas (Chappell et al., 1991). AT1 receptor and AT2 receptor immunoreactivity was previously localized to pancreatic acinar cells in mice and rats, with staining intensity being much weaker than that observed in the pancreatic duct cells of the exocrine pancreas (Leung et al.,

Fig. 6.1 A proposed existence and functional implications of a pancreatic acinar cell RAS in the pancreas

6.2

Ductal Cell RAS

93

1997a). In addition, angiotensin II immunoreactivity was localized predominantly to ductal epithelium and there was less pronounced immunoreactivity in the acinar cells of mouse exocrine pancreas (Leung et al., 1998). On the other hand, RT-PCR experiments revealed angiotensinogen, AT1 , and AT2 receptors in isolated rat pancreatic acinar cells (Tsang et al., 2004). In that study, exogenous addition of angiotensin II stimulated a dose-dependent secretion of digestive enzymes from acinar cells (Tsang et al., 2004), which is consistent with findings in pancreatic acinar AR42J cells (Cheung et al., 1999). In the human pancreas, angiotensinogen and AT1 immunoreactivities were localized in the ductal epithelium of exocrine pancreas; the precise localization of RAS components in acinar cells have yet to be determined (Lam & Leung, 2002). As far as clinical setting is concerned, acinar RAS components are sensitive to pathophysiological conditions, such as pancreatitis and hypoxia (Leung & Chappell, 2003). Figure 6.1 presents a schematic representation proposing the existence of acinar RAS and its potential functional correlates in the exocrine pancreas. Further discussion on the research progress on the RAS in exocrine pancreas concerning pancreatic inflammation will be critically reviewed in Chapter 8.

6.2 Ductal Cell RAS The pancreatic duct cell is a minor cell type in the exocrine pancreas, comprising only a small portion of the organ: 14% by volume in human, 4% in guinea pig, and 2% in rat (Githens, 1988). The duct cell has three primary functions: (1) it provides a structural scaffold for acinar cells; (2) it facilitates the flow of digestive enzymes to the duodenum; and (3) it produces sodium bicarbonate for the neutralization of gastric chyme. Defects in ductal function may be attributed to various pancreatic diseases, such as cystic fibrosis, duodenal ulcer, pancreatitis, and pancreatic cancer (more than 90% are ductal in origin). Previous studies have reported the expression and localization of major RAS components in pancreatic duct cells, which may be relevant for the physiology and pathophysiology of the exocrine pancreatic duct system. Firstly, angiotensinogen, the mandatory component of a local RAS, has been demonstrated to be expressed and localized predominantly in the epithelial cells of pancreatic ducts, as well as in the endothelial lining of pancreatic blood vessels (Leung et al., 1999). Interestingly, angiotensinogen was also detected in significant amounts in pancreatic juice from rats; in contrast, rat duct cells did not express angiotensinogen at an immunoassay detectable level. Instead, most of the angiotensinogen-immunoreactive cells were from glucagon-secreting cells (Regoli et al., 2003). In the human pancreas, immunoreactivity of angiotensinogen was found in the cytoplasm of the epithelial cells from pancreatic ductal epithelium, and was upregulated in pancreatic endocrine tumour tissue (Lam & Leung, 2002). The presence of angiotensinogen in duct cells indicates that locally produced angiotensin II should be present in the pancreatic duct. Indeed, angiotensin II immunoreactivity was observed in the ductal epithelial

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cells of medium-sized pancreatic ducts throughout mouse pancreatic tissue (Leung et al., 1998). Moreover, 125 I-labeled angiotensin II binding was observed in canine pancreatic duct tissue (Chappell et al., 1991), with AT2 receptors being the predominant receptors (Chappell et al., 1992). In murines, both AT1 and AT2 receptors were predominantly localized in the ductal epithelium (Leung et al., 1997a). In human, AT1 receptor was also expressed in the pancreatic ducts (Lam & Leung, 2002); ACE expression was detected in the connective tissues surrounding the pancreatic ducts and was co-localized with AT1 receptor in human pancreas (Arafat et al., 2007). Furthermore, several RAS components are consistently observed in pancreatic duct cell lines. For example, CFPAC-1 cells, commonly used in cystic fibrosis studies, were found to express AT1 receptors, thus implicating its role for angiotensin IIinduced ductal ion transport by the duct cells (Chan et al., 1997; Cheng et al., 1999). AT1 receptors have also been found in the cell membranes and, occasionally, the cytoplasm at various levels of expression in several pancreatic ductal adenocarcinoma cell lines; their expression was sensitive to regulation by angiotensin II via mediation of the AT1 receptor (Anandanadesan et al., 2008). As far as functional correlate is concerned, previous studies have suggested a novel role for angiotensin II in the regulation of ion transport from the secretory epithelia of a number of tissues and organs such as the intestine (Cox et al., 1987), trachea (Norris et al., 1991), and epididymis (Leung et al., 1997b). In the intestine, angiotensin II was recently shown to have a direct role in regulating intestinal epithelial Na+ transport via enhanced activity of an intestinal Na+ –H+ exchanger, and thereby controlling electrolyte and fluid absorption (Musch et al., 2009). In the pancreas, ductal RAS has also been shown to have a physiological role in the regulation of ductal cell anion secretion from the pancreatic duct, such that the balance between RAS stimulation and inhibition governs pancreatic ductal HCO3 – secretion. In this regard, angiotensin II was found to induce changes in intracellular Ca2+ and cAMP, thus stimulating Cl– secretion in the cystic fibrosis pancreatic duct cell line CFPAC-1 (Cheng et al., 1999). Selective antagonism of AT1 receptors in CFPAC-1 cells by losartan inhibited Cl− secretion across a CFPAC-1 monolayer. Interestingly, this AT1 receptor-mediated anion secretion was polarity specific, dependent on the application of losartan to the apical or basolateral membranes. These data are consistent with an immunohistochemical study showing differential expression of AT1 receptors in apical and basolateral membranes of mouse pancreatic duct (Chan et al., 1997). Taken together, these data demonstrate that angiotensin II can stimulate Cl− secretion in human duct cells by activating apical Cl− channels via AT1 receptors in a manner that depends on Ca2+ and cAMP (Cheng et al., 1999). On the other hand, it has been reported that AT1 receptor activation-induced increases in calcium affect calcium-mediated chloride channels in dog pancreatic duct epithelial cells and CFPAC-1 cells, further suggesting that angiotensin II may play a role in regulating pancreatic ductal secretion (Fink et al., 2002). Interestingly, angiotensin II can enhance secretin-induced pancreatic secretion while the ACE inhibitor, captopril, can inhibit this pancreatic bicarbonate output from the pancreas (Howard-McNatt & Fink, 2002).

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Ductal Cell RAS

95

About 95% of exocrine pancreatic cancer cases involve adenocarcinomas originating from the pancreatic ducts. Pancreatic ductal adenocarcinoma (PDA) accounts for more than 20% of the mortality rate of gastrointestinal malignancies (Korc, 2003). It is known that vascular endothelial growth factor (VEGF) is an important pro-angiogenic mediator for PDA; it is implicated in tumour progression and resistance, enhancing proliferation and survival of tumour cells (Korc, 2003). In this context, ACE and AT1 receptor were localized to ductal epithelial cells in PDA specimens. Angiotensin II is a potent stimulator of VEGF expression in PDA and this effect is directly mediated by AT1 receptors, which in turn are dependent on tyrosine kinase activity and ERK1/2 MAP kinase activation; in this case, AT1 receptor blocker and ACE inhibitor could decrease VEGF mRNA and protein expression and thus inhibit proliferation of PDA tumour cells (Anandanadesan et al., 2008). Moreover, co-localization of the AT1 receptor and VEGF was observed in ductal cells of normal human pancreas, indicating that there may be paracrine and/or autocrine actions of angiotensin II and VEGF on angiogenesis. Furthermore, angiotensin II is also a potential stimulus for monocyte chemoattractant protein-1 (MCP-1) in PDA cells mediated by AT1 receptor-ERK1/2-NFkB dependent mechanism (Chehl et al., 2009). All these data suggest that RAS antagonism may help reduce tumour growth, angiogenesis and metastasis (Fujita et al., 2002). In summary, Fig. 6.2 provides a schematic representation proposing the existence of ductal RAS and its potential functional correlates in the pancreas.

Fig. 6.2 A proposed existence and functional implications of a ductal cell RAS in the pancreas

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6.3 Stellate Cell RAS Like hepatic stellate cells, pancreatic stellate cells (PSCs) are vitamin A-storing cells and they contribute to fibrosis, a condition characterized by an excess deposition of extracellular matrix proteins (Kordes et al., 2009). PSCs are located at the base of pancreatic acini. They exhibit a radial pattern of cytoplasmic extension, encircling the basal aspect of acinar cells (Omary et al., 2007). In healthy pancreas, PSCs exist in a quiescent state, characterized by the presence of vitamin A-containing lipid droplets in their cytoplasm. In diseased states and under stimulation of growth factors, cytokines and oxidative stress, PSCs undergo proliferation, become responsive to proliferative and profibrogenic factors, and transform into a myofibroblast-like phenotype in which they express α-smooth muscle actin (α-SMA) and synthesize excess amounts of extracellular matrix (ECM) proteins, such as collagen and fibronectin (Apte et al., 1998; Shimizu, 2008). In fact, they are the major source of the ECM that contributes to fibrous tissue during pancreatic injury, notably pancreatic fibrosis, chronic pancreatitis and pancreatic cancers, as evidenced by previous studies (Apte et al., 1999; Masamune et al., 2009). In view of this unique property of PSCs, it is crucial to investigate some novel candidates that may be able to regulate the conversion of quiescent PSCs into activated PSCs. This line of work may lead to the development of new treatments for the management of pancreatic inflammation (Omary et al., 2007). Among these potential candidates, the RAS in the PSCs is emerging as a key mediator which is involved in the regulation of pancreatic fibrosis. The expression and localization of RAS components in PSCs along with the potential role of a local RAS in pancreatic fibrogenesis are discussed henceforth. Stellate cells in the liver, called hepatic stellate cells (HSCs), are known to play a critical role in liver fibrosis. Previous studies have shown that the RAS is a critical player of hepatic fibrogenesis during liver injury (Wynn, 2008). Activated HSCs were found to produce angiotensin II, thus inducing contraction and proliferation of HSCs (Bataller et al., 2000). Meanwhile, RAS inhibition through AT1 receptor antagonism was shown to attenuate liver fibrosis in both in vivo and in vitro studies (Jonsson et al., 2001; Bataller et al., 2000). In addition, angiotensin II and AT1 receptor in lung stellate (Wang et al., 1999) and kidney stellate cells (Kida et al., 2007) have also been implicated in lung fibrosis and renal fibrosis, respectively. In the pancreas, PSCs are subjected to regulation by various endocrine, paracrine and autocrine factors, in particular, the RAS. In this context, PSCs have local RAS components including the AT1 receptor, the antagonism with losartan of which plays a role in the development of pancreatic fibrogenesis (Liu et al., 2005; Nagashio et al., 2004; Reinehr et al., 2004). In situ hybridization and immunocytochemical experiments have shown that expression of AT1 receptor mRNA and protein are localized mainly to cell membrane in isolated human PSCs; however, no angiotensin II was detected in culture media or cell homogenate of isolated human PSCs using radioimmunoassay, suggesting that angiotensin II utilizes paracrine, but not autocrine, signalling pathway via AT1 receptors in PSCs (Liu et al., 2005). mRNA expression of several RAS components (angiotensinogen, ACE, AT1a , AT1b and AT2 ) was also detected in isolated rat PSCs (Hama et al., 2004). Meanwhile,

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AT1 receptor protein expression was confirmed in PSCs which are upregulated by exposure to high glucose concentrations (Ko et al., 2006); additionally, α-SMA- and AT1 receptor-positive PSCs have been observed in mice (Nagashio et al., 2004). Converging evidence of the presence of a stellate cell RAS indicates its clinical relevance to pancreatic fibrogenesis such as pancreatitis and pancreatic cancer. RAS activation has been shown to play important roles in various tissue fibrosis and inflammation processes in RAS blockade studies (Schieffer et al., 1994, Ishidoya et al., 1995, Yoshiji et al., 2001). Locally produced angiotensin II promotes cell inflammation, ECM protein secretion, and collagen degradation (Ko et al., 2006). Angiotensin II could enhance DNA synthesis in PSCs by over-expression of Smad7 via the protein kinase C pathway (Hama et al., 2006). Angiotensin II could also enhance DNA synthesis by activating ERK through EGF receptor trans-activation, thus triggering the proliferation of activated PSCs (Hama et al., 2006), a key event in pancreatic fibrosis. Thus, inappropriate PSC activation via angiotensin II-mediated signalling in chronic pancreatitis and pancreatic cancer represents a potential target for intervention, such as by RAS blockade (Omary et al., 2007). Indeed, RAS inhibition could, by influencing PSC activation and proliferation, attenuate pancreatic inflammation and fibrosis (Kuno et al., 2003; Yamada et al., 2003). In experiments using an animal model of T2DM (Otsuka Long Evans Tokushima fatty rats), markers of islet fibrosis were decreased following application of an ACE inhibitor, and α-SMA positive PSCs and pancreatic ECM production were significantly reduced following ramipril treatment (Yoshikawa et al., 2002; Ko et al., 2004). In isolated PSCs exposed to high glucose levels for 24 h, AT1a receptor subtype and ACE mRNA expression were upregulated without any change in expression of AT1b receptor subtype and AT2 receptors while angiotensin II production was increased. Interestingly, ACE inhibition and AT1 receptor antagonism improved ECM production and islet fibrosis in PSCs under high glucose exposure (Ko et al., 2006). Furthermore, AT1a receptor knockout (−/−) mice were reported to have reduced PSC activation relative to wild-type mice. When pancreatic fibrosis was induced by repeated episodes of cerulean injection-induced acute pancreatitis, pancreatic fibrosis was attenuated in AT1a receptor (−/−) mice compared with that in wildtype mice (Nagashio et al., 2004). In light of these findings, these data indicate that the AT1 receptor plays an important role in the development of pancreatic fibrosis through PSC activation and proliferation. In summary, the potential functional relevance of the pancreatic stellate cell RAS to fibrosis is depicted in Fig. 6.3.

6.4 Islet Cell RAS Only 1–2% of the mass of pancreatic cells are of endocrine origin, namely the Islets of Langerhans. Among the five cell types found in islets (see Chapter 1, Section 1.1), the β-cell is the major cell type. The β-cell produces the peptide hormone insulin in response to nutrients (the primary stimulus) and hormonal and neural regulation, and plays a critical role in the maintenance of glucose homeostasis. Other peptides and proteins have recently been suggested to have profound impacts on pancreatic

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Fig. 6.3 A proposed existence and functional implications of a stellate cell RAS in the pancreas

islet secretion, differentiation, and growth as well as insulin sensitivity, and thus glucose metabolism and diabetes mellitus pathology (Leung & De Gasparo, 2009). Among these peptide candidates, the RAS has emerged as a potentially important regulator of islet physiology and pathophysiology (Leung, 2007). The expression, localization, regulation, and clinical relevance for T2DM of a pancreatic islet RAS have been critically reviewed in detail elsewhere (Leung & Carlsson, 2005; Leung, 2007; Chu & Leung, 2009). In the following section, solid evidence for the presence of several key RAS components in different islet cell types from the pancreas, and their potential roles in islet β-cell function and dysfunction from our laboratory and others, are discussed below. (Pro)renin mRNA was previously localized to reticular fibers within pancreatic islets and endothelial cells of the pancreatic vasculature using in situ hybridization,

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while (pro)renin protein was localized predominantly to the β-cells themselves in human pancreas (Tahmasebi et al., 1999). In view of this observation, the authenticity of locally expressed (pro)renin within islets remained in question as (pro)renin could potentially be synthesized in the islet interstitium and taken up by the islets themself (Sealey et al., 1996; Tahmasebi et al., 1999). In term of angiotensinogen the mandatory component of a local RAS, researchers had been unable to detect its mRNA expression in rat pancreas for some time, perhaps due partly to technical limitations of mRNA assays, i.e. low detection sensitivity, as well as mRNA degradation during its extraction from the RNase-rich pancreas (Campbell & Habener, 1986). Recently, however, angiotensinogen mRNA was unequivocally identified in isolated mouse pancreatic islets using quantitative RT-PCR, confirming the existence of an islet RAS in the pancreas (Lau et al., 2004). Meanwhile angiotensinogen expression at both mRNA and protein levels in β-cells were shown to be markedly upregulated in transplanted islets and islets in diabetic mice (Lau et al., 2004; Chu et al., 2006). Interestingly, another recent study demonstrated expression and localization of angiotensinogen in islet α-cells of the rat pancreas (Regoli et al., 2003). In this study, immunoreactivity for angiotensinogen was detected at the periphery of the islets and most of the angiotensinogen-immunoreactive cells were predominantly glucagon-positive, a property consistent with α-cell identity. It is thus suggested that angiotensinogen and glucagon are secreted by different pathways in α-cells: glucagon is secreted through a regulated pathway, while angiotensinogen is produced through a constitutive pathway (Sernia, 1995; Regoli et al., 2003). Meanwhile, ACE and ACE2 mRNA and protein were also found in rat pancreatic β-cells, localized mainly to the microvasculature and the islet periphery. Concurrently, ACE and ACE2 expression in islets were also found to be upregulated in the Zucker diabetic fatty rat, a rat model of obesity-induced T2DM (Tikellis et al., 2004). In the human pancreas, expression of both AT1 and AT2 receptors have been identified in pancreatic islets, with AT1 receptors being located mainly in cells near the centre of the islets and co-localized with insulin secreting β-cells (Tahmasebi et al., 1999). In the mouse pancreas, AT1 and AT2 receptor transcription and translation products were found in isolated mouse pancreatic islets; immunocytochemical data further confirmed that AT1 receptor immunoreactivity was co-localized with that for insulin in islet β-cells (Lau et al., 2004). In addition, AT1 receptor protein formation was found to be increased in transplanted islets (Lau et al., 2004) and islets from obesity-induced db/db mice of T2DM (Chu et al., 2006). In contrast, AT2 receptor expression was predominantly co-localized with somatostatin secreting δ-cells in the periphery of rat islets (Wong et al., 2004). In keeping with these findings in rat pancreas, abundant expression of AT2 receptors was also detected in two pancreatic endocrine cell lines, namely RIN-m and RIN-14B, which may be involved in the regulation of somatostatin release (Wong et al., 2004). In another pancreatic β-cell line, called INS-1E, AT1 receptor immunoreactivity was found to be localized specifically on the cell membrane; when the INS-1E cells were exposed to high glucose concentrations, it resulted in dose-dependent upregulation of AT1 receptor

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Table 6.2 The expression and localization of key RAS components from different cell types of pancreatic islets RAS components

Cell types in pancreatic islets

References

Renin/pro-renin

β cells and Islet vasculature

Angiotensinogen

α cells and β cells

ACE

Islet microvasculature

ACE2 AT1 receptor

β cells and islet icrovasculature β cells

AT2 receptor

δ cells β cells

Tahmasebi et al. (1999) and Tikellis et al. (2004) Lau et al. (2004), Regoli et al. (2003) and Chu et al. (2006) Lau et al. (2004) and Chu et al. (2006) Tikellis et al. (2004) Tahmasebi et al. (1999), Lau et al. (2004), Ko et al. (2004) and Chu et al. (2006) Wong et al. (2004) Chu et al. (2010)

mRNA and protein expression (Leung & Leung, 2008). Taken together, key localization and expression studies of major RAS components in different cell types of the pancreatic islets are summarized in Table 6.2. The aforementioned findings provide substantial evidence in favour of the existence of an islet RAS in the endocrine pancreas. The immediate question that needs to be raised is: what are the functional and clinical implications of an islet RAS? In this context, islet blood flow, which plays a key role in controlling islet secretion, is subjected to regulation by islet RAS activity. Angiotensin II, a potent vasoconstrictor, was demonstrated to reduce islet blood flow while RAS blockade with either ACE inhibitor or AT1 receptor blocker, enhanced islet blood flow in rat perfusion studies (Carlsson et al., 1998). Consistently, the first phase of glucose-stimulated insulin release was impaired in animals subjected to acute angiotensin II treatment, and this effect was reversed by administration of an ACE inhibitor, an effect related to islet blood flow (Pollare et al., 1989). Furthermore, subsequent studies have shown that AT1 receptor blockade can enhance islet blood and insulin secretion, thus improving glucose tolerance and glycemic control in rat (Huang et al., 2007). Besides being a potent vasoconstrictor on the islet blood flow thus insulin release, angiotensin II can also influence directly islet cell secretory function which is independent of its pancreatic blood flow. To address this issue, isolated islets devoid of vasculature were employed in our laboratory. Intriguingly, our results showed that angiotensin II dose-dependently inhibited insulin release in response to glucose stimulation, an effect that was completely rescued by AT1 receptor antagonism (with losartan) in mouse isolated pancreatic islets. The regulatory mechanism by which angiotensin II induces inhibitory effects on glucose-stimulated insulin secretion may be due, at least in part, to decreased (pro)insulin biosynthesis rather than an effect on glucose oxidation rate (Lau et al., 2004). On the other hand, losartan selectively improved glucose-stimulated insulin secretion and (pro)insulin biosynthesis, and thus reduced hyperglycemia and glucose intolerance using a mouse model of T2DM

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(Chu et al., 2006). Furthermore, the up-regulated NAD(P)H oxidase expression and activation were also mediated by islet AT1 receptor (Hirata et al., 2009) while specific antagonism was shown to be beneficial for islet cell secretion and β-cell mass, probably as a result of effects on oxidative stress-induced islet apoptosis and proliferation (Chu & Leung, 2007; Cheng et al., 2008), AT1 receptor-ERK1/2 dependent mechanism was potentially involved in the angiotensin II induced islet MCP-1 up-regulation, the co-localization of islet local ACE and MCP-1 indicated the endogenous interaction between islet RAS and islet inflammation (Chipitsyna et al., 2007). Taken these data together, all available findings indicate that islet cell secretory function and cell mass may be subjected to regulation by the RAS involving primarily the AT1 receptor via three potential mechanistic pathways: islet blood flow, (pro)insulin biosynthesis, as well as oxidative stress and inflammation (Fig. 6.4). Further discussion on the research progress on the RAS in the endocrine pancreas concerning T2DM will be critically reviewed in Chapter 8.

Fig. 6.4 A proposed existence and functional implications of an islet cell RAS in the pancreas

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Pollare T, Lithell H and Berne C. A comparison of the effects of hydrochlorothiazide and captopril on glucose and lipid metabolism in patients with hypertension. N Engl J Med 321:868–873, 1989. Regoli M, Bendayan M, Fonzi L, Sernia C and Bertelli E. Angiotensinogen localization and secretion in the rat pancreas. J Endocrinol 179:81–89, 2003. Reinehr R, Zoller S, Klonowski-Stumpe H, Kordes C and Haussinger D. Effects of angiotensin II on rat pancreatic stellate cells. Pancreas 28:129–137, 2004. Schieffer B, Wirger A, Meybrunn M, Seitz S, Holtz J, Riede UN and Drexler H. Comparative effects of chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on cardiac remodeling after myocardial infarction in the rat. Circulation 89: 2273–2282, 1994. Sealey JE, Catanzaro DF, Lavin TN, Gahnem F, Pitarresi T, Hu LF and Laragh JH. Specific prorenin/renin binding (ProBP). Identification and characterization of a novel membrane site. Am J Hypertens 9:491–502, 1996. Sernia C. Location and secretion of brain angiotensinogen. Regul Pept 57:1–18, 1995. Shimizu K. Mechanisms of pancreatic fibrosis and applications to the treatment of chronic pancreatitis. J Gastroenterol 43:823–832, 2008. Tahmasebi M, Puddefoot J, Inwang E and Vinson G. The tissue renin-angiotensin system in human pancreas. J Endocrinol 161:317–322, 1999. Tikellis C, Wookey P, Candido R, Andrikopoulos S, Thomas M and Cooper M. Improved islet morphology after blockade of the renin-angiotensin system in the ZDF rat. Diabetes 53: 989–997, 2004. Tsang SW, Cheng HK, Leung PS. The role of the pancreatic renin-angiotensin system in acinar digestive enzyme secretion and in acute pancreatitis. Regul Pept 119:213–219, 2004. Wang R, Ramos C, Joshi I, Zagariya A, Pardo A, Selman M and Uhal BD. Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides. Am J Physiol 277:L1158–L1164, 1999. Wong P, Lee S and Cheung W. Immunohistochemical colocalization of type II angiotensin receptors with somatostatin in rat pancreas. Regul Pept 117:195–205, 2004. Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 214:199–210, 2008. Yamada T, Kuno A, Masuda K, Ogawa K, Sogawa M, Nakamura S, Ando T, Sano H, Nakazawa T, Ohara H, Nomura T, Joh T and Itoh M. Candesartan, an angiotensin II receptor antagonist, suppresses pancreatic inflammation and fibrosis in rats. J Pharmacol Exp Ther 307:17–23, 2003. Yin G, Yan C and Berk BC. Angiotensin II signaling pathways mediated by tyrosine kinases. Int J Biochem Cell Biol 35:780–783, 2003. Yoshiji H, Kuriyama S, Yoshii J, Ikenaka Y, Noguchi R, Nakatani T, Tsujinoue H and Fukui H. Angiotensin-II type 1 receptor interaction is a major regulator for liver fibrosis development in rats. Hepatology 34:745–750, 2001. Yoshikawa H, Kihara Y, Taguchi M, Yamaguchi T, Nakamura H and Otsuki M. Role of TGF-beta1 in the development of pancreatic fibrosis in Otsuka Long–Evans Tokushima Fatty rats. Am J Physiol Gastrointest Liver Physiol 282:G549–G558, 2002.

Part III

Research Progress of the RAS in Pancreas

Chapter 7

Basic Techniques for Pancreatic Research

7.1 Cell Models Cell culture system studies complement animal studies, providing an indispensable tool for examining the underlying signalling mechanisms by which certain physiological responses are evoked and, at the same time, allowing observation to be attributed to distinct individual treatment factor. They are often applied as primary screenings of the potential effects of certain drug treatment prior to further animal studies. Experiments utilizing pancreatic cell lines or primary pancreatic cell cultures have been widely reported. The term cell line generally refers to an immortalized group of cells originally derived from a single clone which can be, in principle, maintained permanently and proliferated upon exposure to appropriate medium in vitro. Many cell lines are derived from abnormal tissues, such as cancer specimens or tumour cells. The pancreatic cell lines that are most commonly used in experiments, along with some pertinent information and relevant literature for each cell line model, are discussed and summarized in this Chapter (see Table 7.1). Pancreatic cancer cell lines are divided into exocrine (adenocarcinoma) and endocrine tumor classes. It is worth noting that a primary culture is different from a pure cell line; primary culture refers to cells or tissues that are taken directly from the source tissue specimens. Primary cultures generally include populations of cells. Protocols for the isolation and culture of individual cell type components (e.g., acinar and islet cells) from the pancreas are well established in the field and are discussed briefly in this chapter. An important advance in cell culture is the development of the co-culture system. Under certain conditions, different cells are grown together in a mixture; and in some protocols, the cells are separated while allowing the flow of secretory molecules through a permeable membrane insert. The choice for the membrane inserts with an appropriate pore size is critical to best suit for different experimental needs. A large pore size allows not only the exchange of secretory molecules but also the migration of cultured cells between the two compartments. In the context of pancreatic cancer research, such application becomes a useful technique in studying the metastatic properties of the cells under different microenvironment (Chen et al., 2009). Local invasion of cells can often be directly captured using P.S. Leung, The Renin-Angiotensin System: Current Research Progress in The Pancreas, Advances in Experimental Medicine and Biology 690, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9060-7_7, 

109

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7 Basic Techniques for Pancreatic Research Table 7.1 The common cell lines used in basic pancreatic research

Disease of origin Exocrine tumor (Adenocarcinoma)

Species Acinar cell tumor Ductal/epithelial cell tumor

Endocrine tumor

Glucagonoma Insulinoma

a b

Mouse Rat Mouse Rat Human

Names

266-6 AR42J LTPA ARIP PL45 Capan-2 PANC-1 Capan-1a SW-1990b Mouse αTC1 Rat AN 697 Mouse β-TC-6 MIN6 Rat RIN-m5F INS-1 Hamster HIT-T15 Human NES2Y

References Siveke et al. (2008) Chan & Leung (2009) Leiter et al. (1978) Wang et al. (2001) Suemizu et al. (2007) Shi et al. (2008) Lau et al. (2008)

Chuang et al. (2008) Petersen et al. (2000) Mwangi et al. (2008) Kaneko et al. (2009) Onoue et al. (2008) Leung & Leung (2008) Mariogo et al. (2009) Ou et al. (2005)

Cells derived from liver metastasis site. Cells derived from spleen metastasis site.

conventional microscopes (Farrow et al., 2009). The use of a co-culture system reveals the importance of cell-to-cell interactions. In this context, a recent report elegantly revealed, using a rotational co-culture system that the pancreatic duct-derived epithelial cells assist in maintaining the structural integrity and functional viability of isolated human islets (Murray et al., 2009). Co-culture paradigms are also commonly utilized in pancreatic stem cell research wherein the stem cells are grown and developed under the influence of specified factors secreted by another cell type within the co-culture system. One prominent application in the field is exemplified by the maintenance of embryonic stem cells on a feeder cell layer (Zhou et al., 2008). Apart from culturing pancreatic cell lines or isolated primary cell culture, the whole pancreas can also be isolated and cultured under suitable conditions (Parsa & Marsh, 1976). This organ culture technique is commonly seen in studying embryonic pancreas which serves as a good platform to reveal its developmental changes. A suitable culturing medium supplemented with enough growth factors is required so that the pancreas will be developed to mimic the actual developmental scenario in the embryos. Typically, the dissected pancreatic rudiments from rodent and/or human embryos are laid on the permeable membrane inserts at the air-liquid interface and the culture can be sustained for about one week (Guillemain et al., 2007; Castaing et al., 2005). Some studies have also reported with the use of the hanging drop technique when culturing the embryonic pancreas (Tei et al., 2005).

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7.2 Animal Models The use of animal models is very useful in the development of novel therapeutic strategies and therefore the creation of experimental models is of major emphasis on our current research. Various animal models are available to represent the clinically relevant conditions of Type 1 and Type 2 diabetes mellitus (T1DM and T2DM, respectively). Figure 7.1 and Table 7.2 provide a summary of rodent models of T1DM and T2DM which are commonly employed in contemporary research on diabetes.

Fig. 7.1 A summary of the common experimental models of T1DM in basic pancreatic research. NOD, Non-obese diabetic; BB, Bio-breeding; GAD, glutamic acid decarboxylase

Table 7.2 The common rodent models of T2DM and their characteristics in basic pancreatic research (the table is modified from Masiello, 2006, Kadowaki, 2000)

Knockout mouse models

Rat models

Obesity

Associated insulin resistance

db/db

Yes

Yes

PDX-1 +/– IRS-2 +/– GLUT4 –/– ZDF rat GK rat n-STZ rat

No

No

No No

Yes Yes

Severe diabetes at adult age Impaired glucose tolerance Mild to severe diabetes Not diabetic

Yes No No

Yes No No

Mild diabetes at adult age Mild diabetes at adult age Mild hyperglycemia

Phenotype

PDX-1, pancreatic duodenal homeobox factor 1; IRS-2, insulin receptor substrate-2; Glut4, solute carrier family 2 (facilitated glucose transporter), member 4; ZDF, zucker diabetic fatty; GK, Goto-Kakizaki; n-STZ, netonatal streptozotocin.

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7.2.1 Animal Models of T1DM Typical T1DM animal models include Non-obese Diabetic (NOD) mice and Biobreeding (BB) rats. The genetic backgrounds of both rodents were designed specifically to contain many genes related to susceptibility to autoimmunity, thus producing spontaneous induction of T1DM in the adult animals (von Herrath & Nepom, 2009). These experimental T1DM models provide a good platform for basic scientists and clinicians so as to facilitate research aimed at elucidating the mechanistic pathways involved in diabetes onset and thus informing potential avenues of prevention. The presence of some specific cytokines was recently demonstrated to be necessary for the development of T1DM (Sutherland et al., 2009). Only subtle differences in T1DM onset have been identified between NOD mouse and BB rat studies. Briefly, there appears to be no sex difference in T1DM onset in BB rats (Verheul et al., 1986); meanwhile female NOD mice often become permanently hyperglycemic at an earlier age and at a higher frequency than male NOD mice (Atkinson & Leiter, 1999; Leiter et al., 1987). Chemical induction strategies are also commonly employed to generate animal models of T1DM. Early studies used alloxan, a glucose analogue that is toxic to β-cells, to produce experimental diabetes (Lenzen & Panten, 1988). More recently, streptozotocin (STZ) has been purported as a replacement chemical for alloxan. STZ has been widely utilized to induce T1DM because of its greater selectivity for β-cells and longer systemic half-life (Junod et al., 1969). STZ is a naturally occurring chemical that is specifically destructive to the insulin-producing β-cells in vivo. It is structurally similar to glucose, and thus can be transported into the β-cells through GLUT-2, but not other types of glucose transporters (Schnedl et al., 1994). There are two major methods for STZ-induced diabetes in rodents, including a single high dose administration (150 mg/kg) (Suen et al., 2006) and a multiple low dose administration (70 mg/kg/day) for consecutive 5 days (Kroon et al., 2008). Experimental findings have shown subtle differences in the dynamics of diabetes formation by these two modes of injection although there is a general observable phenomenon that animals receiving a high-dose STZ administration do not display a higher mortality rate. A multiple low-dose administration method has also been reported to generate a better animal model of possessing progression and long-term complications of diabetes (Arora et al., 2009). Apart from the typical use of STZ to generate T1DM animal models, other study has reported the incorporation of glutamic acid decarboxylase (GAD)-reactive T-cells into NOD mice so as to trigger the attack of the host’s islet cells and thus accelerates the onset of diabetes (Zekzer et al., 1998).

7.2.2 Animal Models of T2DM Various T2DM animal models which mimic the disease are also well established (LeRoith & Gavrilova, 2006; Cefalu, 2006). A key clinical characteristic of patients with T2DM is the presence of peripheral insulin resistance. Thus knockout mice

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Animal Models

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have been bred to develop insulin resistance and T2DM. Numerous knockout mice models of T2DM have been developed including, but name a few, knockout of genes for the following proteins: insulin receptor substrate-2, GLUT-4, phosphoinositide3 kinase, and peroxisome proliferator–activated receptor γ. Knockout of such genes interferes with normal insulin signalling, thus influencing glucose homeostasis or the normal cellular glucose metabolism (Kadowaki, 2000; Masiello, 2006; Brissova et al., 2002; Katz et al., 1995). A number of transgenic T2DM models are involved in the knockout of phenotypic factors along the pancreatic development. Pancreatic duodenal homeobox factor 1 (PDX-1)-knockout mice is one of the common seen T2DM animal models which falls into this category. A heterozygous mutation of the PDX-1 gene is linked with one of the commonly known maturity onset diabetes of the young (MODY). As PDX-1 is a critical transcription factor in governing proper pancreatic β-cell differentiation as well as maintaining a normal β-cell phenotype, including its growth and secretory functions, a deficiency of this factor will result in a significant reduction of β-cell mass and an impaired insulin-secretory response upon glucose challenge (Hart et al., 2000). Of particular interest in this context is the genetically modified db/db mouse, frequently used in T2DM research, which has had its lep genes knocked out. The resultant absence of leptin disrupts normal appetite regulation and energy intake. As adults, db/db mice become obese and develop obesity-induced T2DM. Such animal model is often associated with features manifested in typical patients with T2DM, including neuropathy and cardioregulatory dysfunctions, which enables it to be suitable for studying these diabetic complications (Goncalves et al., 2009). The Zucker Diabetic Fatty (ZDF) rat has also been used as representative model for T2DM in rats. Genetic predisposition or general phenotype of ZDF rats than makes them a good model (Finegood et al., 2001). This diabetes-prone strain of animals, also with defects in their leptin receptors, has an obviously enhanced β-cell apoptosis that could possibly be attributed to lipotoxicity (Shimabukuro et al., 1998). Different with this obese ZDF rat, another common model, the GotoKakizaki (GK) rat, is a lean model of T2DM. These rats are characterized by a defective β-cell mass when young who progressively result in a markedly impaired glucose tolerance at the adult stage (Briaud et al., 2002). A high-calorie diet is commonly administered in order to induce the diabetic condition; in some cases, low doses of STZ treatment are used to achieve similar results without rendering any significant damages to islet morphology (Okamoto et al., 2008). STZ can also be used to induce T2DM in neonatal rodents. In this method, a mild and stable form of T2DM is produced by a single dose of STZ (90 mg/kg) in 2-day-old neonatal mice or rats. This approach can induce a limited degree of β-cell damage followed by limited regeneration of islet cells as the animals grow, ultimately resulting in β-cell secretory dysfunction in the adult animals (BonnevieNielsen et al., 1981). This can be a good model for studying what the regenerative mechanisms of the defective pancreas are and how the promotion of β-cell neogeneis is like by activating the undifferentiated progenitors. Diabetes models have also been developed in fish. The zebrafish, in particular, has been widely used in pancreatic development and regeneration research owing to its amenability to direct

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observation of its embryonic development under a microscope (Kinkel & Prince, 2009). Additionally, research in the dogfish has yielded some novel dogfish-derived peptides reported to have anti-diabetic effects (Huang & Wu, 2005).

7.2.3 Animal Models of Pancreatitis There are both surgical and non-surgical methods by which to produce experimental models of acute pancreatitis (AP). A critical appraisal of recent advances in basic research in AP can be found in our recent review article (Chan & Leung, 2007). Figure 7.2 summarizes the most commonly used animal models of AP. Non-surgical, or drug induction, methods include, but are not limited to, caeruleinarginine-, and choline-deficient ethionine diet-induced approaches. Among them, a peptide analogue of the digestive enzyme cholecystokinin (CCK), called caerulein has been frequently used to induce AP in experimental animals. Two intra-peritoneal injections of a supraphysiological dose of caerulein are able to achieve a mild form of AP while multiple doses of caerulein can induce a severe form (Chan & Leung, 2006). The success rate of caerulein-induced AP in terms of degree of pancreatic injury and damage is normally assessed in terms of elevation of plasma α-amylase and lipase levels, as well as through evaluation of pancreatic edema and pancreatic histopathology, hours after administration of caerulein. Surgical induction methods include, to name but a few, vascular-induced AP, duct obstruction-induced AP, closed duodenal loop-induced AP, and duct infusioninduced AP. The duct obstruction-induced model is especially common in AP research, requiring the relatively simple procedure of double ligation of the common biliopancreatic duct near the duodenal wall (Chan & Leung, 2007). This model

Fig. 7.2 A summary of the common experimental models of acute pancreatitis (AP) in basic pancreatic research

7.3

Islet and Acinar Cell Isolation

115

focuses on the etiology of AP as it mimics gallstone obstruction-induced pancreatitis in the clinical setting. It is interesting to note that the pancreatitis condition in this model could be completely reversed by removal of the blockade after a 24-h occlusion period (Azima et al., 1996). Simply put, its advantages have made this model a favourite for the studies of AP physiology and pathophysiology as well as studies of therapeutic treatments for the clinically relevant condition of gallstone-induced AP.

7.2.4 Animal Models of Pancreatic Cancer Nude mice have been an important xenograft model for studying human pancreatic cancers in terms of their development, metastasis or response to different pharamacological agents (Shi & Xie, 2000). Both the subcutatneous injection of a pancreatic cancer cell suspension and, more commonly, the surgical orthotopic implantation of human pancreatic tumour fragments into the mouse pancreas, have been applied in many pancreatic cancer studies (Kim et al., 2008; Chen et al., 2009; Pérez-Torras et al., 2008). The latter model allows spontaneous widespread metastasis of the cancer cells to occur within a short time period, though the metastasis potential is often cell line dependent (Garofalo et al., 1993). Incorporation of green fluorescent protein into cancer cells or tumours can facilitate real-time visual imaging and assessment of tumour growth in vivo. There are also many laboratory carcinogens available to chemically induce pancreatic cancers in animals. The organ specific carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), has been used extensively to induce pancreatic adenocarcinomas by implanting it directly into the pancreas of Sprague-Dawley rats (Longnecker, 1990). While DMBA-induction method may require a longer period for tumourigenesis to occur, is the resultant tumours are relatively accessible for growth examination during drug treatment compared with tumours generated in surgical induction models.

7.3 Islet and Acinar Cell Isolation A substantial advancement in the pancreatic research is owed to the development of islet and acinar cell isolation methods. The isolation of islets, in particular, greatly facilitates ex vivo functional studies, allowing more direct in vivo translation compared other β-cell line studies. Recently, substantial efforts have been put into harvesting islets with enhanced improvement in purity, viability and functionality. Optimization of the conditions for enzymatic digestion of pancreatic tissues often dictates the success of the isolation process. The typical procedures for islet isolation are schematically illustrated in Fig. 7.3. There have been numerous reports comparing the isolation outcomes achieved using different collagenases with various proteases (Brandhorst et al., 2003, 2005). A typical digestion protocol calls for intra-ductal administration of collagenase so as to achieve a more thorough infusion and digestion process; however, the conditions for such digestion need

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Fig. 7.3 A schematic diagram illustrating the basic experimental procedures for islet isolation

further optimization to prevent intra-islet penetration of the enzymes (Cross et al., 2008). Supplementary reagents (i.e., Ficoll or Histopaque) have been included to separate cell types, greatly facilitating islet isolation in digested tissues by density gradient (Anderson et al., 2007; Ohgawara et al., 1998). Despite this, complete removal of the peri-acinar fibrous tissue remains the limiting factor for harvesting of islets. Various islet purification strategies have been applied. For example, dithiozone, an established dye for chelating zinc in islets, can produce a red-purplish colour to assist in the identification of the islets (McNary, 1954). Some new digestion chambers have also been developed to assist in mechanical dissociation or filtering out of unwanted components (Gray et al., 2004; Dufrane et al., 2005). In addition

7.4

Islet Transplantation

117

to harvesting islets, recent studies have reported protocols to purify β-cells from islets, based on either β-cell specific molecules (Banerjee & Otonkoski, 2009) or manipulation of their cellular metabolisms followed by flow cytometry (Smelt et al., 2008). The protocols for the pancreatic acinar cell isolation have been well described previously (Schulz et al., 1988). The process appears to be indifferent to which typical enzyme digestion procedure is used. One major obstacle is maintaining the survival of the isolated acinar cells. Decreases in amylase and lipase activity over time in culture are also commonly noted. Optimization of the culture conditions and nutritional set-up is important for maintenance of acinar cell viability for longterm experiments (Kurup & Bhonde, 2002; Singh et al., 2008). Interestingly, several cytokines become upregulated during the isolation of acinar cells from pancreatic tissues via NF-κB and p38 MAP kinase. This observation appears to be due, at least in part, to their blunt response to some other physiological stimulus in culture (Blinman et al., 2000).

7.4 Islet Transplantation The islet transplantation procedure has been a critical breakthrough in treating patients with T1DM and some severe forms of T2DM with chronic complications. The recent success of the Edmonton protocol for islet transplantation has brought about a new era for adequate control of euglycemic status, though insulin independence may not be sustained permanently (Shapiro et al., 2006). Current limitations include the fact that the transplanted islet grafts gradually lose their secretory function and thus may ultimately succumb to failure due to inadequate revascularization or allograft host immune rejection (Rickels et al., 2007). The inner core of the islets, constituted predominantly by insulin-secreting β-cells, is especially susceptible to insufficiency of oxygen and nutrients (Vasir et al., 1998). There have been various strategies developed to maintain β-cell graft function and islet survival in vivo, leading to improvement in islet transplantation (Narang & Mahato, 2006). Different experimental animal models of islet transplantation have been developed as a platform for the assessment of the outcome from various manipulations and monitoring host immune response.

7.4.1 Animal Models of Islet Transplantation Transplantation studies, especially of xenotransplantation (between species), require the use of immunodeficient animals in order to avoid acute graft rejections. The most common host animal model is the nude mouse, a genetic mutant that thus has an inhibited immune system due to its lack of a thymus. Most nude mice, such as BALB/c nude and NU/NU mice, are T-cell deficient, having developed with only a B-cell system. On the other hand, the Severe Combined Immunodeficient (SCID)

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mice have a total immature cellular or humoral immune system. These mice fail to develop any B-cells or T-cells to activate the compliment system. SCID mice are thus widely used as a tool to host transplanted tissues in different studies. The SCID mutation has also been developed on the NOD background, creating the NOD-SCID mouse used in diabetes research. Many researchers choose to perform human islet transplantation studies into the diabetic NOD-SCID mouse model. Some laboratories have even humanized these mice by engraftment of human blood cells (Brands et al., 2008). Unlike other tissue or organ transplantations, islets are not transplanted homotropically into the highly sensitive pancreas, but instead are transplanted into other heterotropic graft sites. Clinical islet transplantation is normally performed through infusion of the isolated islets via the hepatic portal vein into the liver. Yet studies using transplanted mouse models have revealed profound functional impairment in islet grafts retrieved from the liver. These retrieved islets were found to have markedly depleted expression of several β-cell phenotypic factors as well as low insulin release in response to a glucose challenge. These observations have drawn the attention to the notion of implantation site-dependent functional performance of the transplanted islet grafts (Mattsson et al., 2004; Lau et al., 2007). In view of the fact that the loss of islet graft functions might be partly attributable to the necessity for multiple islet donors to cure each individual T1DM patient, researchers are working to find an optimal anatomical site for islet transplantation (van der Windt et al., 2008). New islet transplantation sites, such as the renal capsule, spleen, or the omental pouch, have been reported in animal studies (Merani et al., 2008). The route of each transplantation method offers particular benefits. In the clinical setting, subcutaneous transplantation maximizes patient safety. In our laboratory, we have established the renal capsule as an experimental transplantation site; a brief flow of procedures used in this approach is illustrated in Fig. 7.4. While transplantation studies usually involve intra-species tissue grafting, recent advances have also suggested utilization of islets from xenogenic donors (Schuurman & Pierson, 2008; Cozzi & Bosio, 2008). This idea has been an important breakthrough for dealing with the limited availability of human donor islets. Porcine islets have received substantial attention, particularly since it was demonstrated that they respond to glucose challenge in the same physiological ranges as do human islets (Dufrane & Gianello, 2008). Furthermore, porcine islets may perform better than islets from other species, such as the rat, in terms of achieving hyperglycemia reversal (Davalli et al., 1995).

7.4.2 Recent Advances in Islet Transplantation Minimization of acute rejection of grafted tissues is probably the most critical issue in achieving successful clinical transplantation. This issue is being addressed by studies of immuno-isolation of transplanted islets by microencapsulation. Encapsulation of islets with biomaterials can provide a physical barrier while allowing the passage of glucose and insulin and, at the same time, preventing the

7.4

Islet Transplantation

119

Fig. 7.4 The photographs showing the procedures of islet transplantation in mouse. a The mouse was first anesthetized. b The left abdomen was cut open to expose the left kidney. c A small incision was made on the renal capsule. d Islets were injected between the renal parenchyma and capsule using a positive displacement pipette (withdrawn from Suen, 2007)

entry of large molecules like antibodies (Campos-Lisbôa et al., 2008; Teramura et al., 2007). This strategy is designed to greatly maintain islet graft survival in the host, thus facilitating xenogenic-based therapeutic options. A common protocol with slight variations for islet encapsulation has been reported in many studies (Foster et al., 2007; Calafiore et al., 2006) and the procedures involved are illustrated in Fig. 7.5. Typical protocols make use of the alginate-based materials for microencapsulation, titrated against a cation solution (usually calcium or barium) for gelification and beads formation. Biocompatibility of the encapsulation materials is the major concern for success, and in this context, alginate is a suitable choice for not interfering with the celluar functions of the transplanted islets (Fritschy et al., 1991). Recent findings have even suggested that immunosupression might not be necessary to maintain long-term normoglycemia in diabetic animals given xenogenic islet transplantations (Meyer et al., 2008). These data provide preliminary support to the possibility that immunosuppressive drugs may be minimized after islet transplantation in patients with diabetes.

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Fig. 7.5 A schematic diagram depicting the standard procedures for islet microencapsulation

Various pre-transplantation biological manipulations of the islets have also been suggested to optimize protocols for feasible islet grafting. Gene delivery of the vascular endothelial growth factor (VEGF), for example, in isolated human islets can boost revascularization in transplanted diabetic SCID mice models (Narang et al., 2004); results from glucose tolerance tests also revealed better performance in ameliorating hyperglycemia in animals transplanted with VEGF-transfected islets (Chae et al., 2005). Chemical-induced transfection would generally be preferable to viral vector in gene delivery in clinical applications, as the latter may affect cellular immunogenicity or induce chemokine production.

7.5 Expression and Functional Studies Commonly employed experimental techniques for pancreatic research include, but are not limited to, molecular and functional studies which are summarized in Fig. 7.6.

7.5

Expression and Functional Studies

121

Fig. 7.6 The common experimental techniques recruited in basic pancreatic research

7.5.1 Gene expression studies In terms of molecular techniques, expression studies of a particular gene at the mRNA level are indispensable. Polymerase chain reaction (PCR) is a typical strategy used to compare gene expression levels in a semi-quantitative manner. Quantitative real-time PCR can be applied to measure accumulation of gene products of interest by analysis of a fluorophore during the exponential stages of the PCR. The threshold cycle (CT ), when the amplified target genes are first detected, are compared and the exact fold change between different treatment groups can thus be obtained. Two common dyes used in real-time PCR are Sybr Green, which tags double-stranded DNAs, and the Taqman probe, which releases fluorophores only when a specific gene sequence is amplified. Precise localization of specific genetic transcripts in cells or tissues requires use of in situ hybridization methodology, which directly labels gene transcripts of interest either in tissues sections or in whole mounts by means of complementary nucleotide binding.

7.5.2 Protein Expression Studies Studies of protein expression levels are also indispensable for pancreatic research. Western blotting (Burnette, 1981) employing antibodies for particular proteins of interest is the most common method used to assess protein expression levels. Protocols for the isolation of membrane or nuclear proteins have been well developed for the analysis of numerous proteins. If quantification of a protein or peptide is

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required, the enzyme-linked immunosorbent assay (ELISA) methodology (Lequin, 2005) should be used in which the amount of the target protein or peptide in the sample is proportionally reflected by a colorimetric method. The concept of antigenantibody binding has been further elaborated and applied in a technique named flow cytometry. This fluorescence-based technique is useful in terms of allowing simultaneous detection and quantification of multiple antigens of interest. The data generated, in addition, allow a gated strategy in which a particular portion of cells within a population is analysed. A specialized type of flow cytometry, named the fluorescence-activated cell sorting (FACS), is a powerful technique which allows the purification of a particular cell type of interest within a pool of cell population based on its specialized antigen expression. This has been widely applied in different stem cell research (Alexander et al., 2009). Localization of target proteins within a cell or tissue can be achieved by immunocytochemistry (ICC) or immunohistochemistry (IHC), respectively. Specimens, either paraffin-embedded or cryo-preserved, are fixed and labelled through incubation with specific antibodies against the proteins of interest. Immunoreactivity is reflected in staining or fluorescence intensity, thus allowing an assessment of protein expression levels as well. Co-application of two or more antibodies can be used to demonstrate simultaneous expression of proteins within cells or tissues. Multiple-protein ICC and IHC is best achieved with fluorescent labelling protocols, which requires a fluorescent microscope for detection and imaging. A confocal imaging system further allows three-dimensional reconstruction of tissues, such as whole-mount pancreas tissue. Two-photon microscopy, a viable alternative to traditional confocal microscopy due to its lower phototoxicity and deeper tissue penetration, was recently demonstrated to depict the dynamics of the exocytotic events in a β-cell within a mouse pancreatic islet (Takahashi et al., 2002).

7.5.3 Transfection Genetic modification requires transfection and transduction techniques. These approaches enable foreign nucleic acids to be introduced into host cells through various non-viral and viral vehicles, respectively. Different with employing viral particles as vectors, typical non-viral transfection is a chemical-based method that allows certain nucleic acid sequences to directly enter the cell cytoplasm through alteration of the cell membrane. A commonly used reagent is the Lipofectamine. Most transfected/transduced genes may only be transiently expressed, but some viral vectors (e.g. lentiviruses) allow more stable transduction due to their ability to incorporate genes into the host genome, thus being replicated in host cell mitosis. If a stable and long-term transduction is aimed, the genes are often transduced with a resistance gene to particular antibiotics. Selection of the successfully transduced cells by those antibiotics after the transduction procedures allows a thorough screening and the propagation of the transduced-only cells. The RNA interference (RNAi) technique, especially using short interfering RNAs (siRNAs), is commonly used in gene knockdown experiments. RNAi impedes the expression of a specific

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gene by annealing to their mRNA sequences and thereby marking them for enzymatic degradation. Recent studies also make use of the small-haripin RNA/short hairpin RNA (shRNA) for achieving a gene knockdown. The shRNA sequences contain a tight hairpin turn that allows an increased stability of the structure and

Fig. 7.7 A flow chart showing the general viral-mediated transduction procedures on cultured cells. ∗ A range of multiplicity of infection (MOI; usually 0.5, 1, 2, and 5) should be tested to determine the optimal transduction efficiency. # A killing curve of the antibiotics for each specific cell type should be tested beforehand to determine the minimal amount of antibiotics used to kill all untransduced cells

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achieves a more stable knockdown efficiency. These processes can be done both in vitro in cell culture system and in vivo within targeted organ systems, as recently described in the literature (Larson et al., 2007). A general viral-mediated transduction procedure on cultured cells is illustrated in Fig. 7.7. Verification of a successful transduction experiment is normally done by transducing the green fluorescence protein (GFP)-tagged viruses into cells. Representative images of the human fetal pancreas-derived progenitor cells from our laboratory transduced with the GFP viral particles are shown as an example in Fig. 7.8.

Fig. 7.8 The representative images showing the human fetal pancreas-derived progenitor cells transduced with the green fluorescence protein-tagged lentiviral particles in different multiplicity of infection (MOI). Note that the transduction efficiency (percentage of transduced cells) is different under different MOI. Blue: DAPI; green: viral GFP. Magnification ×630, scale bar =40 µm (For interpretation of the references to colour in this figure legend, please be referred to the online version.)

7.5.4 Functional Studies In vitro (or ex vivo) functional studies, which have frequently been applied in pancreatic research, entail morphological and functional assessments of an isolated islet. This approach allows detailed examination of pancreatic islet function and β-cell structure that may be affected by different risk factors such as glucotoxicity, lipotoxicity, hypoxia or oxidative stress. The physiological response of an islet to glucose-stimulated insulin secretion is often studied as a functional correlate of in

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vivo physiology. Other insulin secretagogues (i.e., like amino acids, glyceraldehyde, and K+ ions) are also commonly used to test islet secretory capacity. ELISA is used to quantify secretion of peptides of interest, such as (pro)insulin or C-peptide, as well as to assess the extent of cell proliferation or cell death, using bromodeoxyuridine and cell death ELISAs, respectively. Since normal glucose-stimulated insulin secretion (Define GSIS) depends on normal functioning of islet ion channels, the patch-clamp technique can also be applied to examine the channel activities, thereby reflecting ion movement dynamics (Marigo et al., 2009). Several typical parameters should be examined and recorded in in vivo functional studies of the pancreas; they include animal survival, body weight, and water consumption. Blood samples are usually collected to determine glucose or insulin levels. The oral glucose tolerance test (OGTT) or intraperitoneal glucose tolerance test (IGTT) are also common methods used to assess the animals’ abilities to reverse hyperglycemia. In these assays, glucose solution is either gavaged or injected, and blood glucose levels are then monitored within 240 min. The animals’ insulin sensitivity can be assessed by an insulin tolerance test wherein insulin is administered intraperitoneally and subsequent changes in blood glucose levels are monitored. Insulin resistance can be measured and quantified using a hyperinsulinemic euglycemic clamp (Wilkes et al., 2009). In this method, insulin is continuously perfused into the animal and the amount of glucose required to compensate for the increased insulin levels without causing hypoglycaemia is carefully examined and recorded. Greater glucose supplementation requirement indicates greater insulin-sensitivity.

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Chapter 8

Current Research of the RAS in Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) accounts for more than 90% of human diabetes and the incidence of new cases is ever increasing worldwide; its prevalence thus places huge demands on healthcare resources and presents a new challenge for the control of disease globally. T2DM is a metabolic disease characterized by hyperglycemia because of enhanced peripheral insulin resistance, impaired β-cell function and decreased β-cell mass. During the development of peripheral insulin resistance leading to an increased demand for insulin secretion, β-cell mass is significantly enhanced by hyperplasia and hypertrophy of β-cells concomitantly with an increase in insulin production as a compensatory mechanism to control normal blood glucose concentrations. As long as β-cell compensatory capacity is maintained, insulin resistance on its own is not sufficient to trigger overt T2DM (Wang & Jin, 2009). In view of this fact, the effective strategy and management of T2DM are to target for the improvement of islet β-cell mass and function rather than peripheral insulin resistance. As of yet, there are still no cures for T2DM and, in view of this fact, basic and clinical research studies are working to elucidate underlying mechanisms and molecules involved in regulating pancreatic islet cell function and T2DM as such findings may produce much needed candidate therapies. This mechanism-driven approach should provide new insights into the development of novel strategies for cost-effective prevention of T2DM and associated metabolic syndrome disorders, and possibly treatment of these conditions as well. There are numerous frontiers in diabetic research dealing with recent developments and new discoveries in the field, including some cutting-edge therapeutic approaches (see articles in special issue by Leung et al., 2006). Many regulators with potential roles concerning β-cell function and insulin resistance remain to be characterized in detail. Among these, there are a number of novel amino acids, peptides and proteins that may affect pancreatic insulin secretion and insulin sensitivity as well as β-cell growth and differentiation (see articles in special issue by Leung & De Gasparo, 2009). Notwithstanding the existence of these promising candidates, there are three physiological regulators, namely angiotensin II, vitamin D and glucagon-like peptide 1 (GLP-1), which are emerging from the growing list of candidates related to islet function and T2DM. In this Chapter, we will provide a critical appraisal of contemporary research progress on the novel

P.S. Leung, The Renin-Angiotensin System: Current Research Progress in The Pancreas, Advances in Experimental Medicine and Biology 690, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-9060-7_8, 

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roles of the renin-angiotensin system (RAS), vitamin D and GLP-1, with particular emphasis on the putative interactions of the pancreatic islet RAS with vitamin D and GLP-1, especially involvement of a RAS-vitamin D-GLP1 axis in the integration of islet function and its clinical relevance to T2DM.

8.1 Basic Studies of the RAS in T2DM Local RASs play a multitude of autocrine, paracrine and/or intracrine roles in the regulation of specific tissue and organ functions (see review by Paul et al., 2006; also see Chapter 5) compared with circulating RASs (see Chapter 4). As discussed in Chapter 6, local pancreatic RASs are now recognized in various cell types of the pancreas including acinar, ductal, stellate and islet cells. The expression of these pancreatic RASs is modulated in response to various physiological and pathophysiological stimuli including, but name a few, hypoxia, islet transplantation, exposure to high glucose concentrations, diabetes mellitus, pancreatitis, and pancreatic cancer. These functional RASs are proposed to have important endocrine and exocrine functions in the pancreas (see review by Leung, 2007a). Of particular interest in this context is the recently identified local RAS in the isolated mouse pancreatic islets which express all major RAS components including angiotensinogen, an obligatory element of a local RAS (Lau et al., 2004).

8.1.1 RAS Blockade Studies in Animal Models of T2DM Such islet RAS is upregulated in animal models of T2DM, such as in obesityinduced db/db mice (Chu et al., 2006) and in Zucker diabetic fatty (ZDF) rats (Tikellis et al., 2004) and, more importantly, blockade of the RAS improves β-cell mass and function in these two animal models of diabetes. Several additional animal models for the study of islet RAS blockade in T2DM have also been developed recently. For example, long-term (24-week) administration of the angiotensin-converting enzyme (ACE) inhibitor ramipril to diabetic Otsuka LongEvans Tokushima fatty (OLETF) rats can prevent islet destruction by fibrosis, as evidenced by the expression profile of TGF-β and its downstream signalling molecules (Ko et al., 2004). Similarly, chronic treatment (10 weeks) with either an ACE inhibitor (perindopril) or type 1 angiotensin II receptor (AT1R) blocker (irbesartan) attenuates islet fibrosis and reduces islet cell apoptosis, and oxidative stress in ZDF rats (Tikellis et al., 2004). On the other hand, hyperglycemia, as observed in T2DM, has recently been shown to activate the RAS in pancreatic islet and stellate cells; RAS blockade ameliorates the angiotensin II-induced pancreatic islet inflammation and fibrosis that is exacerbated by chronic exposure to high glucose concentrations (Lupi et al., 2006; Ko et al., 2006). Consistent with these in vivo findings, exposure of INS-1E cells (a pancreatic β-cell line) to a range of glucose concentrations (0.8, 5.6, 11.1, 28 and 56 mM)

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induces dose-dependent upregulation of AT1 receptor transcription and translation (Fig. 8.1a, b). Such AT1 receptor activation could result in inhibition of glucose-stimulated insulin secretion from the INS-1E cells (Fig. 8.1c). The potential mechanism mediating this effect may be involved in AT1 receptor activation of NADPH oxidase-induced oxidative stress (Leung & Leung, 2008). The data imply that prolonged exposure to high glucose levels (chronic hyperglycemia) is glucotoxic for β-cells such as INS-1E cells, and concomitantly induces AT1 receptor upregulation on these cells, thus leading to impaired insulin secretory function. Taken together, these in vivo and in vitro studies provide a convergence of evidence indicating that pancreatic islet RAS activation may be involved in impaired insulin secretion and enhanced oxidative stress-induced islet cell apoptosis and islet fibrosis; in other words, excessive RAS activation may lead to islet dysfunction and decreased β-cell mass.

Fig. 8.1 Expression of AT1R and insulin release from INS-1E cells in exposure to different concentrations of glucose. a Quantitative PCR analysis. b Western blot analysis. c Glucose-induced insulin secretion from INS-1E cells in response to 5.6 and 28 mM glucose. All data are expressed as mean±SEM; ∗ p < 0.05 vs. 5.6 mM; ∗∗ p < 0.01 vs. 5.6 mM; +p < 0.05 vs. cells exposed to 28 mM glucose; ++p < 0.01 vs. cells exposed to 28 mM glucose (data are extracted from Leung & Leung, 2008)

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8.1.2 RAS Blockade-Induced Protective Mechanism In spite of the aforementioned demonstrations, the precise mechanism(s) by which the local pancreatic RAS modulates islet function remains to be elucidated. The hypothesis that there is a change in AT1 receptor expression in T2DM which enables endogenous levels of angiotensin II to impair islet function has recently been tested in obesity-induced db/db mice (Chu et al., 2006). Though the pancreatic islet AT1 receptor had no obvious effects on normal islet function, it was upregulated in the obesity-induced db/db T2DM mice; this upregulation has negative effects on glucose-stimulated islet insulin secretion and (pro)insulin biosynthesis and on islet blood flow (Chu et al., 2006). These findings provide, at least partly, a novel explanation for the reduced incidence of T2DM that has been observed in a number of clinical trials of AT1 receptor antagonism in individuals at high risk for the disease (Chu et al., 2006; Leung & De Gasparo, 2006). In addition, AT1 receptor antagonism (for 8 weeks) was shown to attenuate NADPH oxidase-induced oxidative stress in isolated islets from db/db mice in vivo; this in turn results in a down-regulation of uncoupling protein 2 (UCP2) expression, which is associated with augmented β-cell insulin secretion and reduced loss of β-cells to apoptosis (Chu & Leung, 2007). In keeping with these findings, chronic AT1 receptor antagonism has been shown to improve islet cell function and structure in OLETF rats and db/db mice, an effect that is apparently mediated by NADPH oxidase-induced oxidative stress (Nakayama et al., 2005; Shao et al., 2006). In light of the above evidence, it is plausible to propose that AT1 receptor activation in T2DM mediates increases in oxidative stress followed by resultant reductions in UCP2 activity which, in turn, leads to pancreatic islet β-cell dysfunction. We have recently shown that angiotensin II can exert a glucose-induced action on Kv currents in isolated mouse islets. In that study, exogenous addition of angiotensin II reduced Kv current amplitude under normal, but not high, glucose conditions. This angiotensin II-induced effect on Kv channel activity was abolished by inhibition of angiotensin II type 2 receptor (AT2 receptor). Our data suggest that hyperglycemia alters β-cell function via mediation of the Kv channel expression and activity, and that this process may be associated with AT2 receptor, but not AT1 receptor (Chu et al., 2010). Nevertheless, the precise mechanism(s) whereby the AT2 receptor modulates channel activity, and thus pancreatic β-cell secretory function, has yet to be fully determined. In summary, basic research studies have garnered convergent data indicating that islet RAS has a novel role in islet function and that its dysregulation may lead to impaired islet function and structure, and ultimately T2DM. In this context, the islet RAS regulates pancreatic islet blood flow, oxygen tension, and islet (pro)insulin biosynthesis (Lau et al., 2004; Kampf et al., 2005; Chu et al., 2006). Meanwhile, it also mediates NADPH oxidase-driven UCP2 activation and thus oxidative stressinduced β-cell apoptosis and fibrosis (Chu & Leung, 2007). Blockade of the islet RAS improves β-cell secretory function, islet oxidative damage and structure, and glucose tolerance in experimental T2DM models (Chu et al., 2006; Chu & Leung, 2007; Cheng et al., 2009). These findings indicate that inhibition of islet RAS activation is an alternative approach to protecting islet cell function and preventing the

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Clinical Studies of the RAS in T2DM

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Fig. 8.2 Summarization of the currently proposed mechanism(s) by which AT1R mediates islet cell function and structure as well as insulin resistance in T2DM

development of overt T2DM (Leung, 2007b). Furthermore, RAS upregulation associated increases in islet oxidative stress and/or reactive oxygen species (ROS) may induce pancreatic β-cell dysfunction and increase insulin resistance in peripheral tissues, thus contributing to development of cardiometabolic syndrome abnormalities (Leung & De Gasparo, 2006). Figure 8.2 summarizes the currently proposed mechanism(s) by which the AT1 receptor may mediate islet cell function and structure as well as insulin resistance.

8.2 Clinical Studies of the RAS in T2DM 8.2.1 Recent Clinical Trials on RAS Blockers Complementary to the basic experimental studies, a host of large clinical trials in patients at high risk for T2DM have reported that RAS blockade delays and/or prevents the new onset of T2DM incidence (Scheen, 2004a, b). These clinical trials include, but are not limited to, Heart Outcomes Prevention Evaluation (HOPE), Losartan for Interventions for Endpoints in Hypertension (LIFE), Nateglimide And Valsartan in Impaired Glucose Tolerance Outcomes Research (NAVIGATOR), Captopril Prevention Project (CAPP), (Prevention of Events with Angiotensin Converting Enzyme inhibition (PEACE), and Renal Outcomes With Telmisartan,

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Ramipril or Both in People at High Vascular Risk (ONTARGET). The conclusion that RAS blockade induces T2DM protective effects is further supported by a meta-analysis of randomized clinical studies (Abuissa et al., 2005; Elliott & Meyer, 2007a, b). For example, a meta-analysis of hypertension treatment trials (Elliott & Meyer, 2007a, b) has suggested that angiotensin receptor blockers (ARBs) are more effective than ACE inhibitors (ACEIs) in reducing the risk of T2DM (Lam & Owen, 2007). Among the clinical trial studies, the PEACE Trial Investigators (2008) study of 8290 patients over a period of 4.8 years in Australia showed that RAS blockade with trandolapril (in subjects with stable coronary disease, preserved cardiac function and virtually normal blood pressure) was associated with a 17% reduction in the incidence of T2DM (HR 0.83 (0.72, 0.96; p = 0.01)), though there was no risk reduction for cardiovascular endpoints and this finding appears to be robust. More recently, the ONTARGET investigators (2008) reported that they found that ACE inhibition (ramipril in 1,412 subjects) and angiotensin receptor blockade (Telmisartan in 1,423 subjects) reduced T2DM risk after 4.8 years to 6.7 and 7.5%, respectively, which is substantially lower than the 34% risk expected from comparable HOPE study data (Yusef et al., 2000). And there is further specific pathophysiological evidence for reduction of insulin resistance and for improvement in insulin secretion with RAS blockade in humans. In view of these substantial clinical data, it is plausible to conclude that blockade of the RAS, in particular with ARBs, may be an efficacious clinical intervention for prevention of T2DM (Kintscher et al., 2008).

8.2.2 DREAM Trial and Its Implications The DREAM trial recently reported that 3 years of treatment with an ACE inhibitor (ramipril) caused an increase in the regression from impaired to normal glucose tolerance (p = 0.01; DREAM Trial Investigators, 2006). However, contrary to the HOPE study, they did not find evidence of a reduction in T2DM risk. These negative findings may be attributable to study limitations, in particular the premature termination of the study after 3 rather than 6 years, The DREAM trial was stopped early because the rosiglitazone arm had shown reduction in T2DM risk. Alternatively, the negative findings may be related to the treatment of normotensive rather than hypertensive subjects and the assumption that the two treatment arms of the planned crossover study would work in comparable ways. Hence the subjects were less RAS active than subjects in trials with hypertensive patients, thus suggesting that longer periods of RAS blockade would be indispensable for detection of reduced T2DM risk in DREAM subjects. Regardless, the observation that ramipril did not ameliorate T2DM in the DREAM study did cast some doubt on the underlying mechanism(s) by which RAS blockade is protective against T2DM. Importantly, however, the parallel ramipril arm in this 2×2 factorial study had shown improvement in impaired glucose tolerance (IGT)/impaired fasting glycemia (IFG) regression at 3 years (p = 0.01). IGT is generally accepted to carry a 25% risk

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Current Research on the RAS-Vitamin D-T2DM Axis

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of progression to T2DM over 5 years in white subjects, thus suggesting that RAS blockade with ramipril could therefore be expected to reduce T2DM risk in the long term. Other factors likely to have reduced the power of the shortened ramipril arm of the DREAM study to detect reductions in T2DM risk include the absence of hypertension or of advanced pre-diabetes in recruited subjects. It is important note that the benefits of RAS blockers and thiazolidones may not be simply additive, thus further under powering the ACE inhibitor arm of the 2×2 factorial DREAM study. Finally, the fact that the thiazolidone treatment (rosiglitazone) was associated with increased risk of heart failure in the DREAM study means that this agent is unlikely to be suitable for use at the population level. The evidence to date, therefore, suggests that there is currently a need (i) to establish whether there are mechanisms by which RAS blockade could reasonably be expected to reduce T2DM risks as evidenced by proof-of-concept studies, and (ii) to determine the value of RAS blockade for T2DM risk reduction in healthy subjects. In light of the basic research and clinical data discussed above, it is reasonable to postulate that the protective effects against glycemia induced by RAS blockade involve the local pancreatic islet RAS. The proposed mechanisms whereby RAS inhibition is protective against T2DM include improved pancreatic islet cell function and structure mediated through enhancement of islet blood flow and (pro)insulin biosynthesis with concomitant reductions in ROS-induced apoptosis and fibrosis and reduced peripheral tissue insulin sensitivity mediated through an increase in blood flow of skeletal muscle.

8.3 Current Research on the RAS-Vitamin D-T2DM Axis Vitamin D is best known for its involvement in the maintenance of normal calcium balance. However, it has other functions, and one of these is the recently proposed notion that normal insulin release from the pancreas needs vitamin D in addition to a sufficient supply of calcium. Therefore, pancreas malfunction, such as occurs in T2DM, may have originate partly as a result of vitamin D deficiency, which is a fairly common problem. In support of this view are experimental and clinical data showing that vitamin D deficiency impairs insulin secretion and increases insulin resistance, two typical markers of increased risks of developing T2DM.

8.3.1 Vitamin D and RAS in T2DM Recent data have shown a link between vitamin D and negative RAS regulation. In this context, identification of receptors for 1α,25(OH)2 D3 (1,25-dihydroxyvitamin D3 , the VDR) and of 1α-OHase vitamin D activating enzyme in many tissues (e.g. immune cells, the vasculature, pancreatic islet β-cells) has led to the recognition of many novel roles for vitamin D, in addition to its classical roles in calcium and bone homeostasis (Holick, 2003; Mathieu et al., 1994; Hewison et al., 2007).

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1α,25(OH)2 D3 binding to the nuclear VDR and to VDR in plasma membrane caveolae generates slow genomic and rapid non-genomic responses, respectively (Norman, 2006). In particular, stimulation of islet insulin secretion by activated vitamin D is largely a rapid non-genomic effect following activation of the caveolaeassociated cell membrane VDR by 1,25-dihydroxyvitamin D (Kajikawa et al., 1999). Interestingly, a review of the literature shows that islet β-cell insulin secretion is reduced in hypovitaminosis D and increases, sometimes to normal, with replacement of vitamin D in whole animals, isolated islets, and humans (Mathieu et al., 2005; Bourlon et al., 1999; Boucher et al., 1995). First and second phase of glucose-stimulated islet insulin secretion, impaired in early and sustained vitamin D deficiency respectively, are restored by adequate dietary supplementation with vitamin D, in vitro and in vivo (Kadowski & Norman, 1984; Cade & Norman, 1986). Figure 8.3 depicts a schematic representation of the mechanism by which vitamin D interacts with caveolae-bound and nucleus-bound VDR mediated transduction pathways in the regulation of pancreatic β-cell function. In keeping with these findings, clinical studies have shown an increased prevalence of T2DM in association with vitamin D-deficiency in humans (Scragg, 1981;

Fig. 8.3 A schematic representation of “rapid” non-genomic and “slow” genomic responses mediated by VDR located on the caveola and cell nucleus in the pancreatic beta cells

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Current Research on the RAS-Vitamin D-T2DM Axis

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Scragg et al., 1990, 2004; Forman et al., 2007). Hypovitaminosis D is also associated with metabolic syndrome defining abnormalities and with increased risk of both T2DM and cardiovascular disease (Boucher, 1998; Chiu et al., 2004; Mathieu et al., 2005). Additional supportive data show an association of reduced ultraviolet-B availability with increased risk of T2DM, possibly attributable to altered immunobiology (Staples et al., 2003). Nevertheless, the mechanisms by which vitamin D contributes to the maintenance of normal islet cell physiology remain obscure. Proposed pathways include increasing intracellular calcium ion concentration, which is necessary for activation of some proteases that release insulin from (pro)insulin (Davidson et al., 1988; Sergeev & Rhoten, 1995). Furthermore, vitamin D status has direct associations with insulin secretory function in humans (Clark et al., 1987; Ishida & Norman, 1988; Billaudel et al., 1989; Lee et al., 1994; Boucher et al., 1995; Bland et al., 2004), which can be explained by the fact that circulating 25-hydroxyvitamin D3 is activated to form 1α,25(OH)2 D3 in the islets, which themselves express the VDR. Hypovitaminosis D is associated with elevated blood pressure in humans (Scragg et al., 2007; Chiu et al., 2004) and vitamin D has been shown, experimentally, to reduce renal RAS activity through suppression of renin secretion, specifically (Li et al., 2004), providing a likely mechanism for blood pressure reductions reported with vitamin D supplementation in humans, especially those with hypovitaminosis D (Lind et al., 1988, 1989). Relevant to these effects, it is noteworthy that RAS activity is several folds greater in VDR-null mice than wild-type mice (Li et al., 2002, 2004; Kong & Li, 2003). Furthermore, there is substantial data from both basic and clinical studies demonstrating that increases in pancreatic RAS activity reduce islet β-cell function and structure, whilst increasing the activity of pro-inflammatory factor genes and proteins in experimental animal models of T2DM.

8.3.2 Role of Vitamin D in Modulating Islet RAS Expression and Function In view of this convergence of evidence, we hypothesize that vitamin D regulates pancreatic islet function (insulin secretion) indirectly by modulating pancreatic islet RAS expression and activity, thereby supporting β-cell function. We are now testing this hypothesis in both in vitro and in vivo models of vitamin D deficiency and repletion and in T2DM; this work should provide a proof-of-principle that vitamin D modulates islet RAS activity as well as reducing circulating RAS activity, which in turn regulates islet β-cell function. Interestingly, we have collected data (unpublished) showing that the expression of major RAS components in isolated pancreatic islets were upregulated under a high glucose condition (28 mM) compared to that under a low glucose condition (5.6 mM). For example, AT1 receptor translation and transcription were markedly increased in the high glucose condition while co-exposure of the islets to active vitamin D (calcitriol, most notably at 10–9 M) down-regulated AT1 receptor expression under high glucose concentration

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